MICROFICHE REFERENCE LIBRARY

AT MICROFICHEREFERENCELIBRARYA project of Volunteers in AsiaA Medical Laboratorv for Develoninu Countriaby: Maurice KingPublished by:Oxford University Press/East & Central AfricaP.O. Box 72532Nairobi, KenyaPaper copies are $22.50.Available from:Oxford University Press16-00 Pollitt DriveFair Lawn, NJ 07410 USAReproduced by permission of Oxford UniversityPress, East and Central Africa.Reproduction of this microfiche document in anyform is subject to the same restrictions as thoseof the original document.

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This book aims to bring the mini~mum levelof pathological services within the range ofeveryone in developing countries and iswritten especially for laboratory andmedical assistants who work in healthcentres and district hospitals. Each piece ofequipment needed in a medical laboratoryis fully described and illustrated in detaileddrawings. Every step in the examinationof specimens is simply explained andthe method of performing it illustrated ; themethods chosen are those that give thegreatest diagnostic value at the minimumcost. Ways of obtaining specimens aregiven, and where it might prove helpfulsome anatomy, physiology and a briefaccount of treatment is included. The lastchapter contains a detailed equipment list.This book goes a long way towards defininga complete ‘health case package’ inan important and neglected field.i,. -..1 netOxfordUniversityPressISBN 0 19 264910 8

OXFORD MEDICAL PUBLICATIONSA Medical Laboratoryfor Developing Countries

To all those who might soeasily be diagnosed andtreated if only someone knew how.

A Medical Laboratoryfor Developing CountriesMAURICEM.D. (Cantab.),KINGF.R.C.P. (Lond.1WHO Staff Member, the Lembaga Kesehatam Nasional, Surabaya, Indonesia.Late& Professor of Social Medicine in the University ofZambia and Visiting Professor in Johns Hopkins UniversityLONDONOXFORD UNIVERSITY PRESSDELHI KUALA LUMPUR1973

Oxford University Press, E& House, London W.1GLASGOW NEW YORK TORONTO MELBOURNE WELLINGTONCAPE TOWN IBADAN NAIBOBI DAB ES SALAAM LUSAKA ADDIS ABABADELHI BOMBAY CAI.CuTfA MADRAS KARACHI LAHOBB DACCAKUALA LUMPUR SINGAPORE HONG KONG TOKYOHardbound edition ISBN 0 19 2649 16 7Paperbound edition ISBN 0 19 264910 80 Oxford University Press, 1973AN rights reserved. No part of this publication may be reproduced,stored in a retrieval system, or transmitted, in any form or by any means,electronic, mechanical, photocopying, recording or otherwise, withoutthe prior permission of Oxford University PressFilmset in Photon Times IOpt. byRichard Clay (The Chaucer Press), Ltd., Bungay, Suflolkand printed in Great Britain byFletcher & Son, Ltd., Norwich

First Preface in Standard EnglishThis book has a critical purpose. It aims to bring a minimum level of pathological serviceswithin the range of evevone in the developing countries. It is thus firstly for the laboratoryassistants and medical assistants who work in health centres and district hospitals, for it is theywho must investigate and treat such common and important conditions as anaemia, malaria,leprosy, tuberculosis, trypanosomiasis, and a variety of helm&h infestations. Unless suchdiagnoses as these are routinely confirmed in a laboratory, the medical care that is provided forthe millions who suffer from them must inevitably be inadequate.But it is not enough to know how to take and examine a skin scraping for leprosy. All thenecessary equipment, most of which is very cheap, has to be available-the mere provision of amicroscope alone is not enough. Hence the last chapter contains a list of everything that ahealth centre laboratory requires, so that it can be inserted in a medical stores catalogue andpacked as a complete kit that nzuy be obtainable through UNICEF.If the doctors of the developing countries are to rise to their challenge, which is to care forall the people, they must lead and teach the other members of the health team. Medical studentsmust thus also become expert in the methods described here, so that they can both do themthemselves, and later hand on their skills to others; for these same methods are required inward side rooms, in the consulting rooms of general practitioners, and also in the laboratorythat should be an integral part of every outpatient department.Many readers will not have had much education; so this text has been made as complete as itcan be, and written with a strictly limited vocabulary in the simplest possible way. A count of5,000 words chosen from randomly distributed sections showed only 550 different ones, and it\ is probable that its entire vocabulary contains less than fifteen hundred. Even so it is hoped thatthe more learned reader will not be offended by its style. If he will only bear with the way inwhich it had to be written, he may be able to make good use of what it has to say.Laboratories have to be numerous and cheap if every anaemic child is to have his stoolexamined for hookworms, when there is perhaps only about a dollar a year to be spent onhealth services of ail kinds. The methods have therefore been chosen to be of the greatestdiagnostic value for the limited funds available; the total cost of the equipment in the basic listgiven here being about $500, including the microscope. This then is a medical laboratory at thelevel of an ‘intermediate technology’, which is in sharp contrast to the costly and increasingly

PrefaceThere is believed to be a need for a work of this kind, and if the present printing sells out, itis hoped that an improved second edition will follow. The writer will therefore be pleased tohear from anyone with criticisms or suggestions for improvements.* Colour transparencies forfurther plates will be particularly welcome.Many kind helpers have commented on the experimental edition of this manual, of which5,000 copies were duplicated before this version went to press, and it is not possible to mentionall of them; but especial gratitude is due to Dr. Felicity Savage, now my wife, Ken Lewis, SisterDarrah Degnan, and to Doctors David Morley, Staniey Browne, and A. H. Van Soest, as wellas to Gordon MacGregor, Wilfred de Souza, and Lionel Billows, from whom I hope I havelearnt ‘Easy English’. Several technicians have contributed valuable criticism, and particularthanks are due to David Staples, Peter Ward, and especially to Aleco Zangousa, MartynLinton, and George Mwale. I am also indebted to Judith Mitchell for the intelligence anddevotion with which she typed the manuscript and to Peter Cheese for so willingly developingthe transparencies from which the plates were made.+ Lastly, I should like to thank Mr. Johnson, our departmental cleaner at Makerete for hiscare in reading part of the manuscript. It was from him I first learned how difficult was the taskI had so lightheartedly embarked upon.February 7th 1972 MAURICE KING* All correspondence should be addressed to Oxford University Press, 37 Dover Street,London W. I., United Kingdom.

Second Preface in Easy EnglishFor laboratory assistants and medical assistantsI have written this book tirst of all for you. Let me start by telling you why I have written it.When a patient is sick, he comes for help to a doctor or medical assistant. Let us say that hecomes to see a doctor and that you are a laboratory assistant. The doctor must first find outwhy the patient is sick, so he starts by listening to what the patient says-he takes his patient’sI&tory. NW he looks at or examines the patient. From the history and examination it ispossible to fmd out or diagnose what is wrong with most patients. But very often it is notpossible to diagnose a patient by only listening to him and looking at him. A doctor needs thehelp of the X-ray department, where they take special pictures of patients, or of a medicallaboratory. For example, a doctor may think that his patient has got weak (anaemic) blc od. Hewill need a laboratory assistant to Iind out how weak it is and to help him find out why it isweak. I have therefore written this book to tell you about a medical laboratory and how youcan use the equipment in it to help doctors, and medical assistants, to diagnose their patients.Your job is to find things out about patients and to report (tell) what you have found to theperson who is looking after them. If the right diagnosis is made, patients can be given the righttreatment and will get well. The reports that you make are therefore important, and they mustbe right. If you give a wrong report, the wrong diagnosis will be made, the wrong treatment willbe given, and the patient may not get well. Your work is thus very important.I have also written this book for medical assistants who may be working in health centres ontheir own with nobody to help them in the laboratory. If you are a medical assistant you willhave too much to do to be able to spend much time in the laboratory. You should, however,learn how to do the methods described here sell, so that you can do them on some of yourpatients, where they will be of great help in diagnosis and treatment.If you are going to be able to do your job properly, you must have the right equipment andchemicals. If you do not have what you need-ask for it, and, if necessary, go on and onasking. In the end you will probably get what you want. If you do the methods in this book welland give true reports, you will be very useful indeed. You will be a great help to many sickpeople.Yours sincerely,Maurice King

ContentsChapter1. INTRODUCTION1.1 How to use this book. 1.2 Honesty and responsibility. 1.3 Some special words. 1.4Solutions and suspensions. 1.5 Centrifuging and filtering. 1.6 Acids, alkalis, and salts. 1.7 pHand buffers. 1.8 Indicators. 1.9 Cells. 1.10 Proteins and enzymes. 1.11 Micro-organisms. 1.12Parasites, commensals and infection. 1.13 How organisms are named. 1.14 The different kindsof parasites. 1.15 Putrefaction or rotting. 1.16 Stains. 1.17 Serum and plasma. 1.18 Isotonicsolutions. 1.19 Disinfectants and antiseptics. 1.20 Sterilization. 1.21 The pressure cooker.1.22 Using sterile equipment. 1.23 Laboratory infection. 1.24 Controls,Chapter 2. EQUIPMENT AND CHEMICALS2.1 The equipment described. 2.2 Special equipment in the main list. 2.3 Ordinary equipmentin the main list. 2.4 Chemicals. 2.5 The choices. 2.6 Stock and spares.Chapter 3. MAKING THE LABORATORY READY3.1 Benches and shelves. 3.2 Sinks and staining. 3.3 Water. 3.4 Bottled gas. 3.5 The Bunsenburner. 3.6 Electricity. 3.7 Using the battery of a Land-Rover. 3.8 Using a spare car battery.3.9 Making and using Pasteur pipettes. 3.10 The loop. 3.11 Some practical details. 3.12Washing equipment. 3.13 Routine. 3.14 Time and motion study. 3.15 Stains and reagents.3.16 Acid alcohol. 3.17 Barium chloride. 3.18 Benedict’s reagent. 3.19 Brilliant cresyl blue.3.20 and 3.2 1 Buffers. 3.22a ‘Cellophane’ coverslips. 3.22b Carbol fuchsin (hot method). 3.23Carbol fuchsin (cold method). 3.24 Dilute carbol fuchsin. 3.25 Crystal violet. 3.26Dichromate cleaning fluid. 3.27 Ehrlich’s reagent. 3.28 Field’s stain. 3.29 Form01 saline. 3.30Fouchet’s reagent. 3.31a Haemoglobin diluting fluid. 3.31b Drabkin’s solution. 3.32 Lugol’siodine. 3.33 Leishman’s stain. 3.34 Malachite green. 3.35 Methylene blue in acid alcohol. 3.36Occult bllood reagent. 3.37 Pandy’s reagent. 3.38 PAS test strips. 3.39 Rothera’s reagent. 3.40Saline. 3.41 Saturated sodium acetate. 3.42a Sodium citrate solution. 3.42b 20% Sodiumhydroxide. 3.43a Sulphone test papers. 3.43b 3% Sulphosalicylic acid. 3.44 20%Sulphosalicylic acid. 3.45 White cell diluting fluid. 3.46 A health centre laboratory described.Chapter 4. RECORDS AND SPECIMENS4.1 Records and reports. 4.2 Records for health centres and outpatient departments. 4.3Records for hospitals. 4.4 The plus notation. 4.5 Preventing mistakes. 4.6 Specimen

,:Contentscontainers. 4.7 Capillary and venous blood. 4.8 Cross infection and syringe jaundice. 4.9SyringesJand ‘needles and tubes’. 4.10 Sending specimens to a central laboratory.Chapter 5. WEIGHING AND MEAkURING5.1 Weight. 5.2 The Ohaus triple beam balance. 5.7 Pipettes and measuring cylinders. 5.8Perc~cntages and parts. 5.9 Why and how we measure colour. 5.10 The Lovibond comparator.5.11 The Grey wedge photometer. 5.12 Filters for light. 5.13 The Haldaue scale. 5.14 Usingthe Grey wedge photometer. 5.16 The EEL calorimeter. 5.17 Getting the EEL ready. 5.18Learning how the EEL works. 5.19 Standards for the EEL. 5.20 The cyanmethaemoglobinmethod. 5.21a Oxyhaemoglobin methods. 5.21b Using a graph with the EEL. 5.22 When theEEL goes wrong. 5.23 Focusing the bulb of an EEL. 5.24 Changing the bulb in an EEL. 5.25Changing the selenium cell in an EEL.Chapter 6. THE MICROSCOPE6.1 The !lrn. 6.2 How a microscope works. 6.3 The mirror and the condenser. 6.4 Centering thecondenser. 6.5 The filter holder. 6.6 The condenser stop. 6.7 The objectives. 6.8 The eyepiece.6.9 The tube, the coarse and fine adjustments. 6.10 The mechanical stage. 6.11 Lights for themicroscope. 6.12 The Olympus model K microscope. 6.13 Knowing your microscope. 6.14Using your microscope. 6.15 Specimens of poor contrast. 6.16 Troubles with microscopes.6.17 Some ‘Do’s’ and ‘Don’ts’ in microscopy. 6.18 Looking after microscopes in warm, wetcountries.Chapter7. BLOOD7.1 Haemoglobin. 7.2 The haematocrit. 7.3 The MCHC. 7.4 An anaemia chart for the ‘underfivesclinic’. 7.5 Anaemia. 7.6 Iron deficiency anaemia. 7.7 Folic acid deficiency anaemia. 7.8Anaemia caused by protein deficiency. 7.9 Haemolytic anaemias. 7.10 When to measure thehaemog:.>bin. 7.11 The thin blood film. 7.12 Leishman’s method. 7.13 Faults in a thin bloodfilm. 7.14 Normal white blood cells (leucocytes). 7.15 Platelets. 7.16 The white cell percentagesin normal blood. 7.17 How blood cells are formed in the marrow. 7.18 Abnormal cells inthe blood. 7.19 Abnormal red cells. 7.20 Abnormal white cells. 7.21 Some further bloodpictures. 7.22 The differential white cell count. 7.23 Reticulocytes. 7.24 What a haemoglobinopathyis. 7.25 Sickle cells. 7.26 Two solubility methods for haemoglobins A and S. 7.27aSickle-cell anaemia and the sickle-cell trait. 7.27b Thalassaemia. 7.28 A simple guide toanaemia. 7.29 Counting white cells. 7.30 What an abnormal total white cell count means. 7.3 1Why a thick film is so useful. 7.32 Malaria. 7.33 A diagram of the human plasmodia. 7.34 Themeaning of a positive thick film in malaria. 7.35 Relapsing fever. 7.36 Trypanosomiasis. 7.37Filariasis. 7.38 A concentration method. 7.39 The ESR. 7.40 The form01 gel method. 7.4 1 Theserum urea. 7.42 The blood sugar. 7.43 Measuring the plasma acetone with Acetest tablets.Chapter8. URINE8.1 A clean specimen of urine. 8.2 Why we test the urine. 8.3 Testing the urine for sugar andprotein. 8.4 The meaning of proteinuria. 8.5 Routine urine testing. 8.6 Diabetes and the bloodsugar. 8.7 Acetone. 8.8 Jaundice and some tests for bile pigments. 8.9 Testing for INH and

ContentsPAS. 8.10 Testing for sulphones. 8.11 Pus cells in the urine. 8.12 The meaning of pyuria. 8.13Looking at the centrifuged deposit. 8.14 Three kinds of movement. 8.15 Looking for the ovaof Schlstosoma haematobium.Chapter 9. THE CEREBROSPINAL FLUID9.1 Where the cerebrospinal fluid comes from. 9.2 The importance of lumbar puncture. 9.3Diagnosing meningitis. 9.4 Equipment for lumbar puncture. 9.5 Doing a lumbar puncture. 9.6Two specimens of CSF. 9.9 Cells. 9.10 Pandy’s method. 9.11 Stained films. 9.12 A combinedmethod of examining the CSF. 9.13 The CSF protein. 9.14 Trypanosomes. 9.15 Sugar in theCSF. 9.16 Suppurative or bacterial meningitis. 9.17 Virus meningitis. 9.18 Tuberculousmeningitis. 9.19 Head injury. 9.20 Cerebral malaria.Chapter10. STOOLS10.1 Why we examine the stools. 10.2a The saline stool smear. 10.2b The ‘Cellophane’ thicksmear for ova. 10.3 The formal-ether concentration test. 10.4 The ‘Sellotape’ swab forEnterobius. 10.5 Some common ova. 10.6 Some more ova. 10.7 E. histolqtica and E. coli. 10.8Bacillary and amoebic exudates. 10.9 Identifying E. histolytica. 10.10 Giardia lamblia andTrichomonas hominis. 10.11 Occult blood. 10.12 Measuring the pH of a stool and testing forlactose. 10.13 When to examine the stools.Chapter 11. SOME OTHER SPECIMENS11.1 AAFB and the Ziehl-Neelsen method. 11.2 Preventing false positive reports. 11.3 Harmlessmycobacteria. 11.4a Finding cases of tuberculosis. 11.4b Examining the sputum for helminthova. 11.5 Gram’s method. 11.6 Urethral smears for gonococci. 11.7 Some less commonuses of Gram’s method. 11.8 Looking for Trichomonas vaginalis. 11.9 Testing the gastric juicefor free acid. 11.10 Examining the seminal fluid. 11.1 la Classifying leprosy. 11.1 lb The skinscraping. 11.1 lc Nasal smears. 11.1 Id Examining and reporting on smears for Myco. Zeprae.11.12 Lymph node puncture for trypanosomes. 11.13 The rectal snip for Schistosomamansoni. 11.14 The skin snip for Onchocerca volvulus. 11.15 Skin scrapings for fungi.Chapter 12. BLOOD TRANSFUSION12.1 Blood groups and agglutination. 12.2 Transfusion. 12.3 Antisera. 12.4 Washing red cells.12.5 Blood grouping. 12.6 Cross-matching. 12.7 Rhesus grouping. 12.8 Eldon cards. 12.9Equipment. 12.10 Sharpening needles. 12.11 The pilot bottle. 12.12 Taking blood. 12.13 TheUganda mobile team. 12.14 Storing blood. 12. I5 Making blood transfusion safer.Chapter 13. FOR PATHOLOGISTS, STORES OFFICERS, AND MEDICALADMINISTRATORS13.1 A standard manual. 13.2 The scope of this manual. 13.3 The equipment list. 13.4 Buildup peripheral services first. 13.5a Some chemicals and equipment discussed. 13.5b Upgradingperipheral laboratories. 13.5~ Teaching. 13.6 The supply of complete kits by UNICEF. 13.7The addresses of suppliers. 13.8a General stores required. 13.8b Special equipment in themain list. 13.9 Ordinary equipment in the main list, 13.10 Chemicals. 13.1 1 Preparedreagents. 13.12 Choices. 13.13 Choice 1, The M.R.C. Grey wedge photometer. 13.15 Choice

4~.~~,~~;;‘.‘-:‘1:‘,’ .~, ’ ,;,, .;~~~~;:,is:~,“.,:‘;i;.~, .__))( ~,;I ,- .‘, : ,: : ._ ,, ; 1*.ir ,._/ : -4” +;> ,, ; ,, :+. J L ,.$.,?:ir .) .,;.yp,, ; ,, Contshtsz::,p ,! ?,,I ,.,-F$”/_ 2, The EEL calorimeter. 13.16 Choice 3, Silica gel as a desiccant for microscopes. 13.17:Choice 4, Electric centrifuges. 13.18 Choice 5, A Fuchs-Rosenthal counting chamber. 13.19i.,., Choice 6, INH in the urine. 13.20 Choice 7, The cyanmethaemoglobin method, 13.21 Choice8, Sodium azide. 13.22 Choice 9, Ammonia for the oxyhaemoglobin method. 13.24 Choice11, Dichromate clear@ fluid. 13.25 Choice 12, Additional chemicals for preparing certain.’,’reagents. 13.26 Choice 13, ‘Dextrostix’. 13.27 Choice 14, Rothera’s test. 13.28 Choice 15,‘Ictotest’ tablets. 13.29 Choice 16; Tbe ‘Cellophane’ thick smear. 13.30 Choice 17, ‘Labgaz’.13.3 1 Choice 18, This manual. 13.32 A table of choices. 13.33 References.Epilogue_~,.;,:VocabularyIndexPlatesl-9 Thin blood films, Leishman stainedlO- 17 Blood grouping and sickle cells18-29 Malaria thin blood films, Giemsa stained30-41 Malaria blood films, Giemsa stained42-50 Parasites in thick and thin blood films5 I-55 Blood microfilariae56-64 Urinary deposits65-85 Ova in the stools86-95 Protozoa, etc. in the stools96-100 CSF and sputum101-107 Gram’s method and the Ziehl-Neelsen method

How to use this book 1 1.11 1 Introduction1 .l How to use this bookThis is the most important chapter of all. It tells yousome of the things you must know before you can understandthe methods in the rest of the book. First, you mustlearn how to use a book like this. This is more importantthan learning all the methods, because, if you can use thisbook and have it with you, you can easily read how to dothe methods in it. Try, therefore, to get a copy of thisbook for yourself.The first twelve chapters of this book are written ineasy English with as few new words as possible. You willfind all these new words at the back of the book in aspecial list called the vocabulary index. You can readwhat these new words mean, and you will also see whereyou can find out more about them. If there are still wordsyou cannot understand, get a dictionary. A good dictionaryto get is An English-Reader’s Dictionary by A. S.Homby and E. C. Pamwell published by OxfordUniversity Press. Some people are helped if they say thenew words they find to themselves. The important newwords have been written in thick black type like this.Learn these new words very carefully. When you havefinished reading a chapter, look back over it and makesure that you know all the new words.This book is made in parts or sections. Each sectionhas a number which is written with a dot in it. Forexample, this is the first section of the first chapter, andyou will see 1.1 (one point one) written at the top righthand corner of this page. Section 3.12 means the twelfthsection in Chapter Three.The drawings in this book ai-? called figures. Thesefigures also have numbers, but these have a dash (a shortline) in them. Fi)r example, FIGURE 2-6 (two dash six) ist!~ sixth figure in Chapter Two. The coloured picturesat the end of the book are called plates. Some thingsare shown in the black and white figures and in thecoloured plates. When this happens the number of thecoloured plate has been put beside the black and whitepicture.Some sections and figures have a or b after them,13.8a and 13.8b, for example. 13.8b was added to thebook after the numbering was done, but it is just thesame as any other section. Some sections and figureswere taken away after the numbering was done, and youwill find that they have gone. You will, for example, findno Section 5.3. Some people do not like books numberedin sections and like the pages numbered instead. Booksnumbered in sections are, however, much easier tomake.Very often it is not possible to explain everything atonce, and you will have to look at other sections tounderstand things. You are often asked therefore to lookat a section or a figure in another part of the book--besure you do this. You may not always find what you wantat the beginning of a section you turn to; so be sure youlook right through a section when you are looking forsomething. Sometimes you will find the words ‘seebelow’. This means that you can find more about somethingfurther on in that section.This book has been written to teach you. IF YOU ’ARE GOING TO LEARN, YOU MUST DOEXACTLY WHAT THE BOOK SAYS. You will notlearn if you do not do what the book says. For example,if in Chapter Six you read the words, ‘Look carefully atthe Picture C in Figure 6-4 andjnd the iris diaphragm’,YOU MUST LOOK IN FIGURE 6-4 AND FINDTHE IRIS DIAPHRAGM IN PICTURE C. To helpyou do this, instructions like ‘Look’ and ‘Fin8 havesometimes been written like this. These instructions tellyou to DO something. Don’t read any more until youhave done what you have been told.If a piece of equipment is being described in a chapter,try to find it in your laboratory and look at it while youare reading about it. For example, have the Ohausbaiance beside you when you are reading Section 5.5.Have the microscope beside you when you are readingChapter Six.Large pieces of this book are in thick black writingwith the word ‘Method’ on top ,of them. The methodsections give you instructions (orders) which tell youwhat to do. Follow these methods with great care.There are many figures, and they are all drawn in thesam< way. At first you may find them difficult to understand.FIGURE I- 1 will help you. In each figure there areseveral pictures. in FIGURE l-l picture A is a test tube.Picture B is a test t&e full of water. Picture C is a testtube which is being shaken (moved about). In Picture D

! 1. Introductionarrows like this/ you which picture to/ look at nexteach figure has several picturesin it : each of these pictures hasthis is a --Etest tubefull ofwaterthe large circle isa larger picture ofwhat is inside thesmall circle\ Fthis is theend ofa fingerHthis is a slide from on top)~S&----IIpipette-thisthese linesare meantto looklike glasssis liquidin the pipetteI-this is a slide from the sideslides in the equipment list : itis called the ML numberto try to make thecup look roundthis means theend of thepipette is notdrawn in thepicturethis is an obliqueview of a slide\dots for sectionsf12.3 this is the third section in chapter twelve2-6\this is the sixth figure in chapter twodashes for figuresFig. l-1 Understanding diagramsthe Pasteur pipette (see 3.9) is going to be put into a testtube. A long arrow shows that something is being movedsomewhere. A short thick arrow tells you which pictureto look at next. The short thick arrows in FIGURE I- 1 tellyou to look at Picture A, then at Picture B, then atPicture C, and then at Picture D. Some arrows are blackand some are white. They both tell you what picture tolook at next.In some figures part of a piece of equipment is drawnagain larger. For example, the part of the pipette insidethe small circle in Picture E is drawn again larger insidethe big circle in Picture F.Spots are sometimes used to show the shape ofsomething. The spots in Picture G, for example, havebeen used to show that the cup has a round shape.Pictures H, I, J, and K are all pictures of the same glassmicroscope slide. Picture H is drawn looking down fromon top. Picture I is drawn looking from one side. Picture.I is drawn looking from the end of the slide. Picture K isdrawn looking partly from on top, partly from the sideand partly from the end. It is called an oblique view. Inother figures you will see many microscope slides drawnthis way.In some pictures, things are drawn as near as possibleto what they really look like. These pictures aredrawings. Other pictures give only the idea of something.These pictures are diagrams. FIGURE IO-5 is a drawingand FIGURE 10-4 a diagram.To make drawing easier the parts of a figure are sometimesnot drawn to scale. This means that they are notdrawn the right size compared to one another. Forexample, the finger in Picture E, FWIRE I- 1, is much toobig compared to the cup in Pitt ,3. You will soonlearn to understand the figures and u,:.,;t the wrong scaleof what you see.The number beside the picture of a piece of equipmentis its ML or Medical Laboratory number. You will readabout these numbers in Section 2.1. The number 37

Honesty and responsibility 1 1.2beside Picture K, for example, is the ML number formicroscope slides.The cost of equipment is in American dollars whichare written $. There are 100 cents in a dollar. Forexample, a universal container costs about $0.08, whichis eight American cents. This has been done becauseevery country has different money, and it is only possibleto give costs in one kind of money.Here are some more ways in which you can makebetter use of this book.METHODUSING THIS BOOKDon’t learn it by heart.Don’t try to read it from beginning to end all at once.Make good use of the vocabulary index.Don’t read about equipment you do not have or arenot likely to getIf you are new to laboratory work, read Chapter Oneas far as the end of Section 1.20. Read Chapter Two asfar a’s the end of Section 2.4. Read Chapter Three as faras the end of Section 3.15. Only read about the reagentsin Sections 3.16 to 3.45 when you want to make them.Read Section 3.46 carefully. Read those parts ofChapter Fiie which describe the equipment you have.Read all about the microscope in Chapter Six. Then, inthe other chapters, read about the methods that you aregoing to use most often. After you have done this youcan read anything else which interests you.Don’t worry because this book is so big-you willsoon learn to use most of it.1.2 Honesty and responsibilityOn an earlier page you read about how very important itis to be honest-to tell the truth at all times. But thereare other things that you should know about the rightway to work in a laboratory. The easiest way to tell youabout them is ta write them down like this.METHODHONESTY. AND PROFESSIONAL RESPONSIBILITY IN ALABORATORYAlways tell the truth. NEVER guess what the answershould be on a specimen you have not examined. Awrong result may make a patient die. Any laboratoryworker found doing this deserves to lose his job immediately.He cannot be trusted to work in a hospital or ahealth centre.If you are not sure what to report, say you are notsure. If you are not sure about something, go and talk tothe person who is looking after the patient. If you arelooking at a specimen for a doctor, he will think better ofyou for saying you are not sure. If you have forgotten todo something you have been asked to do. say you haveforgotten. If you have made a mistake, say so. The3mistake may be important, and you will be respected fortelling the truth.If you are given so much work that you cannot finishit, say it is too much. Ask which are the most importantspecimens. Do these. If reports on the other specimensare really wanted, keep them, if you can, until the nextday.Doctors often write ‘URGENT’ or ‘VERY URGENT’ or‘IMMEDIATELY’ on a request form. Doctors mean whatthey say, so do what they say.Sometimes one method leads to another. Do thesemethods, even though they are not asked for. If, forexample, you have found that a patient is very aneemic,do the other methods for anaemia, such as looking at ablood film, or measuring the MCHC.Don’t work with broken equipment, such as a brokenmicroscope with which you cannot give the right report.It is better to give no report than a wrong report.Equipment and reagents cost money. Look after allequipment carefully. Try to use the least amount ofa reagent needed by each method, so that none iswasted.All the equipment and chemicals in the laboratorybelong to the hospital or health centre. This includesneedles, tubes, knife blades, syringes, and spirit. Don’ttake them home with you. To do so is to steal them. Doall you can to stop other people stealing equipment.If you cannot go to work because you are ill or forsome other reason, send a message to say so. The personyou work for will then know what has happened.When you do come back to work go immediately to seethe person you work for and tell him why you wereaway.Most people work for somebody else. If you do notagree with the person you work for, you have the right tosee or write to the person above him. If you do this it isonly fair to tell the person you work for first. Manypeople do not understand this. and it can cause muchtrouble.Don’t try to experiment and to make new methods foryourself. This wastes chemicals and equipment and maybe dangerous.If you are a laboratory assistant, don’t think you are adoctor or a medical assistant. You are not. Don’t thereforetreat patients yourseM If patients ask for the resultof a method, don’t tell them, even if the patient is anurse. Laboratory reports are for doctors or medicalassistants only.When sick people are to be cared for. the day’s workdoes nor start with reading the newspaper. Work doesnot end at 4 p.m. or at any other time! Work in a hospitalor health centre only ends when everything that can bedone for the patients has been done. This care and interestin the job, and what it means to the patients, is whatmakes you into a professional person, like a doctor or ajudge.If you have finished looking at the specimens before itis time to go home, don’t do nothing. There is alwayswork to do in a good laboratory. Make up new reagents.

1 1 Introduction.tidy UP, read your textbook, and try to make your laboratorybatter.Many patients cannot be treated until their reportsare back from the laboratory. If these reports are late,patients cannot be treated a& soon as they should be.Patients are often anxious to start treatment, so thatthey can soon go home to their work and their families.Their beds may also be needed for other patients. Tryhard to send al! tzxwts back to the ward the same dayas the specimen was sent to the laboratory.Patients are often worried and anxious. Talk to them,explain things to them. If you have to hurt patients byputting a needle into them, explain what you are goingto do and tell them why. Most important of all. be kindto patients. think what they need and do all you can forthem. Ona day you too may be a patient and you willknow how much this means.If you follow these rules people will rely on you. Theywill think of you as being kind, honest, reliable, adhard-working. To be thought of like this is precious.1.3 Some special wordsMost of the words you will want to know about areexplained in the vocabulary index. Here are some of thedifficuit ones. Several of them are pairs (twos) of wordswith opposite meanings. Some of them can mean severaldifferent things and only some of these meanings aregiven here.Accurateand inaccurateAccurate means exact. For example, to say that there areabout 360 days in a year would be inaccurate. To saythat there are 365 days would be more accurate. Youwill often have to weigh or count things accurately.Acuteand chronicThese words are used. to describe diseases and havespecial meanings. An acute disease, such as pneumonia(a disease of the lungs), is a short-lasting one from which apatient dies or gets better quickly. A chronic disease lastsa long time, and a patient dies or recovers slowly. Tuberculosisand leprosy are chronic diseases.AdjustTo adjust a machine is to alter or fix it so that it worksbetter. If the brakes on your bicycle do not work, theymust be adjusted so that they do work.AverageLet us take an example. Say that there $re ten boys in aclass and that three are aged 12, two are aged 13, two areaged 15, and three aged 16. We find their average age byadding up the ages of all the boys and dividing this sumby the number of boys there are. The ages of all the boysadded together come to 140 years. There are ten boys inthe class, so we divide by ten and the average age is 14years. You may want to give the average answer to amethod, as in Section 5.14. Take several readings and getseveral answers. Add them up and divide by the numberof readings you took. The result will then be the averageanswer. It will usually be the answer you get most often.Ten is a good number of readings to take because itmakes dividing much easier-you just change the placeof the decimal point (see Section 5.2). Five readings willnot take so long to do but division is not so easy. Let ustake an example. Suppose you are measuring the haemoglobinon the Grey wedge photometer and you get fivereadings of 40,4 1,43,43, and 42. Added up these cometo 209. Divide this by five and the average is 41.8, say42. If you cannot understand averages, do not worrytheyare not important for most of the methods in thisbook.CaseIn medicine the word case has a special meaning. Apatient with a disease is often called a case of thatdisease. For example, we talk about a case of tuberculosis,meaning a patient with tuberculosis.Clear. transparent, opaque, and turbidClean water is clear or transparent; that is, we can lookthrough it very easily. We cannot see through milk, andwe say it is opaque. When we can see little particles(small pieces) floating about in liquid (see below) we sayit is turbid. Muddy water is turbid.DetergentA detergent is a very strong soap. ‘Teepol’, ‘Tide’, and‘Surf’ contain detergents.Dischargesor exudatesThese are fluids which come from some part of the body,such as a wound, a diseased place (a lesion), or from theinside of one of the hollow organs of the body, such asthe intestines. Many discharges and exudates contain pus(see Section 7.14).GraduatedThis word can be used in several ways, but when usedhere it does not mean getting a B.A. degree! A ruler isgraduated into inches or centimetres, and the lines on itare called graduation marks. A measuring cylinder isgraduated in millilitres. Graduated therefore meansdivided up by marks or lines into spaces of a special size(such as inches or millilitres) that can be used to measure

Some special words 1 1.3with. A row of graduation marks is a scale. There is ascale on a ruler or a thermometer (see FIGURE 6-3). Asyou have already read, the word scale can also be used tomean the size of the things drawn in a picture. A weighingmachine is also sometimes called a scale.InstrumentAn instrument is a laboratory machine. A balance, amicroscope, and a centrifuge are ail instruments.-,Male and femaleMale means man or belonging to a man. Female meanswoman or belonging to a woman.Matureand immatureLike many words, these two can be used in several ways.As used here, immature means young and not fullygrown. Mature means fully grown. An adult is a fullygrown man or woman. An infant is a young child.Membraneand filmThe word membrane is used for something very thin.Cells are covered by cell membranes. The thin plastic ofa plastic bag could be called a membrane. As used herethe word film meilzs something spread very thinly on aglass slide, such as a blood film or a film of sputum.Normaland abnormalIf something is seen in most healthy people, we say it isnormal. If it is only seen in people who are sick, we say itis abnormal. For example, healthy people have a fewepithelial cells in their urine. We say therefore that it isnormal to 6nd epithelial cells in the urine. Healthypeople do not have protein in their urine. It is thereforeabnormal to find protein in the urine.There can also be normal or abnormal numbers ofsomething. It is normal for a person to have one or twowhite cells in a cubic millimetre of cerebrospinal fluid(CSF). It is abnormal for him to have as many as 100.Percent‘Cent’ means a hundred, so percent means per hundred.A schoolboy who gets 6 1 percent marks of the marks inan examination gets 61 marks in a hundred. A patientwho has a haemoglobin of 5 g percent has five grams ofhaemoglobin in 100 ml of his blood.Positiveand negativeYou will often find the words positive written + andnegative written -. In this book we use the words positiveand negative like this. Say we are looking for proteinin the urine, and we find protein. We say that the test forprotein is positive. If we find no protein in the urine, wesay that the test for protein is negative. A test may beslightly positive or strongly positive. Read about this inSection 4.4. on the ‘plus notation’.PlasticMany things we use every day are made of what is calledplastic, so also is much laboratory equipment. Almost allpens are plastic, so are the soft kinds of buckets andcups. There are many kinds of plastic; some are hardand some are soft. Polythene is a very commonly usedplastic. It is opaque, it bends easily, and it becomes verysoft in boiling water. Perspex is another plastic. It is hardand completely clear (transparent) like glass or water.Polypropylene is a very strong plastic that can be boiledor autoclaved (see Section 4.6).RodYou will often read the word rod in this book. A rod issomething long and thin like a pencil. A shaft is a rodwhich turns.Symptomsand signsA sign is something in a patient that a doctor or medicalassistant sees, hears, feels, or tests for. This may be spots(seen), noises in the chest (heard), a lump (felt), or proteinin the urine (tested for). A symptom is somethingthat a patient complains of, such as pain or a sore throat.TestTo test something is to see if it is there or to see if it isworking. We test a car to see if it is working and we testthe urine to see if there is any sugar in it. The words ‘test’and ‘method’ can sometimes be used in the same way.Typicaland atypicalTypical means what is ordinarily seen. Atypical meansunusual or extraordinary. Men typically have five fingers;they are atypical if they have six. Patients withpneumonia typically have a fever. It is atypical for apatient with pneumonia to have no fever.Verticaland horizontalA high tree stands straight up and is vertical. The surfaceof water is flat or horizontal. Something which is slopingand is neither vertical nor horizontal is said to beoblique.Zero and infinityZero is another name for ‘O’, nought, or nothing. Infinityis the opposite to zero and means the biggest numberthere is.

‘:i-,v,_’1 1 lntrodudtion1.4 Solutions and suspensionsA substance is anything which is the same all throughand which can be divided without being spoiled. Water,sugar, earth, ink, and wood are all substances. A liquid issomething like water or milk which flows and can bespilt. A liquid will fill the bottom of a cup or bottle andwill spread over the floor. As used in this book a fluid isthe same as a liquid. A solid is something like wood orglass which stays in the same shape when it is moved. Apowder is something like earth or sand. A powder is dryand a liquid is wet. Powders are like liquids because theyfill the bottom of whatever they are in. But a powder doesnot flow as easily as a liquid, and if you look at a powdercarefully you will see that it is made of many small solidpieces. Small pieces like this are often called granules, orparticles. If all the pieces of a solid have the same simpleshape we call these pieces crystals. Sugar and salt makecrystals, so do many other chemicals.If a spoonful of sugar is put into a cup of tea the sugarseems to be lost. But we know it is not really lost,because the tea tastes sweet. We say the sugar has dissolvedin the tea. Sweet tea is a solution of sugar in plaintea. If there is much sugar in the tea and the tea is verysweet, the sugar solution is said to he concentrated(strong). If there is little sugar in the tea and the tea is notvery sweet, the solution of sugar in tea is said to he dilute(weak). The concentration of the solution is the amountof sugar that there is in the tea. If there is so much sugarin the tea that it will not dissolve any more, we say thatthe tea is saturated with sugar, or that there is a saturatedsolution of sugar. A solution of salt in water is calledsaline. Something like. salt, which easily dissolves inwater, is said to be soluble in water. Sand does notdissolve in water and is said to be insoluble in water.Many of the methods in this book use solutions ofsolids in liquids. The solutions used in the methods arecalled reagents. Most of the reagents are solutions ofchemicals in water. Most chemicals are powders orliquids that are made specially pure (that is they haveonly one kind of thing in them). Blood, earth, and urinehave many different things in them and are not chemicals.Sugar is one thing only, so is salt. They can bebought in bottles as pure chemicals. When ordinary sugaris bought as a pure chemical, it is given the chemicalname ‘sucrose’, and ordinary salt is called ‘sodiumchloride’. Unlike salt and sugar most chemicals are notused in homes and have not got ordinary names. Forexample, there is no ordinary name for the chemicalcalled sodium citrate.When tea is stirred, tea leaves move in the tea. We saythey are suspended (hanging) in the tea, or that there is asuspension of tea leaves in the tea. But when the tea in theteacup is still the tea leaves soon fall to the bottom of thecup. The tea leaves form a deposit. The tea above theleaves is called the supernataut fluid or the supernatant.One difference therefore between a solution and asuspension is that in a suspension the solid falls to thebottom. In a solution it does not.In Section 8.8 you will read about testing the urine forbilirubin. In this test urine is mixed with a clear solutionof barium chloride. The mixture of urine and bariumchloride goes white (or yellow) and milky. We say aprecipitate has formed. If the mixture is left to stand thiswhite or yellow precipitate will fall to the bottom of thetube. This is what the word precipitate meanssomethingsolid which is made when two chemicals insolution are mixed and which will fall to the bottom if themixture is left to stand. A precipitate can also beremoved by centrifuging or filtering-read about this inthe next section. The words deposit and precipitate areoften used to mean the same thing.When wet clothes are left in the hot sun, the watersoon disappears and goes into the air. We say the waterevaporates. If water is boiled we can see it turning intosteam, and it will evaporate fast. Some liquids like spirit,methyl alcohol, xylene, and especially ether, evaporatemore easily than water. We say they are volatile. Methylalcohol and ether are very volatile. Spirit, methylalcohol, xylene, ether, and petrol will all burn. We saythey are inflammable. Ether is very inflammable indeed.Take great care when you use these chemicals that theydo not light and cause a fire.1.5 Centrifuging and filtering (FIGURE l-2)Blood is a suspension of very small particles called redcells and white cells in a liquid called plasma (see Section1.17). If a few drops of blood are added to a test tube ofsaline they will form a suspension as in Picture A. If thissuspension were left for a day the cells would slowly fallto the bottom of the tube to make a deposit, and leaveclear supernatant saline on top. A deposit with a clearsupematant is shown in Picture E. Blood cells take along time to deposit when they are left to stand. But wecan make blood cells deposit much faster if we makethem spin (move) round very fast. A machine for spinningtubes round fast is called a centrifuge. There is acentrifuge that can be turned by hand in the main equipmentlist. Look for it in FIGURES 2- 1 and 3- 11. PictureB, FIGURE 1-2, shows the parts of a simple centrifuge.The tube to be spun or centrifuged, such as the tube ofblood cells in saline in Picture A, is put into a biggermetal tube called a bucket. These buckets hang in aholder called a trunion. The trunions fit into the head ofthe centrifuge. The head turns round on a shaft. Thecentrifuge drawn in FIGURE 1-2 has two buckets to holdtwo tubes. Many centrifuges have four buckets, and somehave more than four buckets.When the head of a centrifuge turns round the bucketsin their trunions are thrown outwards. When the centrifugeis spinning very fast, the tubes are horizontal as inPicture C. As the centrifuge slows down and stops thebuckets swing down again as in Picture D. If the tube ofred cell suspension is taken out of its bucket, as inPicture E, the cells will be seen as a deposit on thebottom of the tube. There will be supernatant saline on

Centrifuging and filtering 1 1.5/this is a suspensionof red cells in salinethe suspensionis put intoa centrifugethis is thecentrifugespinningA ‘--- - -zY -- -_, e’ _’the buckets swing out asthe centrifuge spinsthis tube has beenDthe centrifugebeen taken out of the truflion has stoppedTUBES NOT BALANCEDTUBESBALANCEDu- - -deposita conicalgraduatedcentrifugetubein a conical centrifugetube all the deposit isgathered in one place1 l-shaftFig. l-2 The centrifugetop. Centrifuging has made the cells deposit in a fewminutes-the cells have been thrown to the bottom of thetube. These cells would have taken many hours todeposit if they were left on their own.If you stop a centrifuge quickly by holding the shaft,the liquid in the tubes is shaken. This often mixes up thedeposit and the supernatant. To stop this mixing, let thecentrifuge stop slowly by itself.There are two ways of taking the supernatant fluidaway from the deposit. A quick and easy way is to pouroff the supematant. But this mixes up the deposit in thebottom part of the supematant. So, when you want totake all the supematant away from the deposit, youshould use a Pasteur pipette (see Section 3.9).The tubes in a centrifuge must balance exactly. Thatis, the tube at one side of the.head of a centrifuge must beexactly as heavy as the tube at the opposite side of thehead. If the tubes do not balance the centrifuge will shake(move about) as it turns. It will make a noise and may bespoiled. In Pictures A to E, FIGLJRE l-2, only one tube ofsuspension is being centrifuged. It is put into the lefthandbucket in Picture B. To balance it a tube filled withthe same amount of water is put into the right-handbucket. Tubes which contain the same amount of liquidwill balance. Tubes which do not contain the sameamount of liquid will not balance. Two badly balancedtubes are shown in Picture F. Two well-balanced tubesare shown in Picture G. Not only must the tubes onopposite sides of the head be equally full, but they mustalso be the same size and weight. In very fast centrifugespairs of opposite tubes have to be weighed on a specialbalance to be sure they are the same weight (the wordbalance can also mean a machine for weighing). In thecentrifuge described in this book it is enough to use tubesof about the same size and weight and to make sure theyare equally full.There are several kinds of centrifuge tube. Picture Hshows a conical (cone-like) graduated centrifuge tube. A

,::,, ,./’ I1 1 Introductioncone is something shaped like the end of a sharp pencil.Conical centrifuge tubes are useful if there is only a verylittle deposit, because all the deposit comes together atthe very bottom of the tube. Graduated centrifuge tubes(tubes with lines or marks on them) are useful for measuring-seeSection 5.7. A tube of this kind is shown inPicture H. Some centrifuges have buckets which arefixed at an angle, as in Picture I. These are anglecentrifuges.Filtering. Centrifuging is only one way of taking awayor separating the particles from a suspension. Anotherway of separating the particles from a suspension is tofilter it. If a suspension of red cells in saline is pouredthrough a special kind of paper, called a filter paper, thesaline will go through and the red cells will be left behindon the paper. The clear saline is called the filtrate. InPicture 3, FIGURE 8-3, you will see the precipitate madeby mixing urine and barium chloride being removed byfiltration. The precipitate is left behind on the filter paperand the clear filtrate is passing through. Remember that afiltrate is the clear liquid made by filtering and that asupernatant is the clear liquid made by centrifuging.Filter papers are circles of a special white paper likeblotting paper. They are sold in boxes of 100 papers andare shown in FIGURE 2-2 (ML 22). A filter paperbecomes soft and easily breaks when it is wet. Filterpapers are therefore always held in a funnel and have tobe folded in the special way shown in Picture B, FIGURE3-8.Filter papers are expensive, so only use them for filtering.Filter papers are not for cleaning benches or makingnotes!Other things can be filtered, besides particlessuspended in liquids. Pieces of coloured glass can beused to filter light. This is described in Section 5.12.1.6 Acids, alkalis, and saltsAs you have just read, the chemical name for the salt weeat is sodium chloride, It is a harmless substance, but itcan be made by mixing one kind of ‘burning’ chemicalcalled hydrochloric acid, with another kind of burningchemical called sodium hydroxide. By burning we do notmean that these chemicals will catch fire like petrol, butthat if they touch your skin or your eyes they will harmyou as if you had been burnt. If therefore you get theseburning chemicals on your skin or into your eyes, washthem off quickly with plenty of water.Hydrochloric acid is an acid, and sodium hydroxide isan alkali. Mixed together in the right amounts, an acidand an alkali make a salt and water. This can be adang!;rous experiment, so don’t try it! Salts are not burningbecause the acid and the alkali have stopped eachother from being able to burn. As we shall see in the nextsection, they are said to have neutralized each other.Let us take another example. The salt called potassiumsulphate can be made by mixing together the rightamounts of sulphuric acid with the alkali called potas-sium hydroxide. There are several acids in the list ofchemicals. Hydrochloric acid, sulphuric acid, and trichloraceticacid are strong acids. Tartaric and acetic acidsare weak acids. Sodium hydroxide and ammonia arestrong alkalis. Sodium carbonate is a weak alkali. Thereare also many salts, such as sodium citrate, potassiumiodide, and ferric (iron) chloride.1.7 pH and buffersWhen a solution of an alkali is slowly added to a solutionof an acid, the alkali will begin to use up the acid to makea salt and water. When only a little acid has been added,there will still be some extra acid in the solution. Thesolution is said to be acid. When all the acid has beenused up there will only be salt and water in the solution.The solution has no extra acid or alkali and is said to beneutral, but if more alkali is added, there will be extraalkali, and the solution is said to be alkaline. The amountof extra acid or alkali is measured by pH. Don’t worryabout what pH stands for. Remember that pH 1 is veryacid and that pH 14 is very alkaline. pH 7 is haif-way inbetween pH 1 and pH 14 and is neutral. Pure water has apH of 7.If acid or alkali is added to an ordinary solution of saltand water, the pH will change, and the solution willbecome more acid or more alkaline. However, there aresome special solutions of salts made from weak acids andalkalis which can use up extra acid or alkali withoutchanging their pH. These special solutions are calledbuffers. Buffers can be made with any pH, and eachbuffer has its own special pH. A buffer is useful forkeeping a solution at this special pH, even when someacid or alkali is added. The buffer in Section 3.2la,which is used for Leishman’s stain, has a pH of 6.8.Because 6.8 is nearly 7, this buffer is nearly neutral andis only very slightly acid. The buffer used for Leishman’sstain is a mixture of two different phosphate salts. It isused for keeping the pH of Leishman’s stain fixed at 6.8during staining.The buffer for gastric washings, which is described inSections 3.20b and 11.4, is used to neutralize thehydrochloric acid in the stomach. By neutralize (makeneutral) we mean that the pH is brought nearer to 7. Thehydrochloric acid in the stomach is very strong and has apH of about 3.5. This strong acid with its low pH wouldkill the Mycobacterium tuberculosis if it were not soonneutralized by a buffer.1.8 IndicatorsIn the list of chemicals there are some small books of aspecial paper called Universal Indicator Test Paper, pH1 to pH 11. If this paper is put in a strong acid, such ashydrochloric acid, it will go red, showing that the pH isabout 1. If the paper is put in a strong alkali, such aspotassium hydroxide, it will go a deep blue, showing that

Cells 1 1.9the pH is about Il. At pH 4 the paper is orange and atpH 8 it is green. By putting a piece of indicator paper ina solution we can easily see what pH the solution is.Paper which changes colour when the pH changes iscalled indicator paper.There are many kinds of indicator paper. Most ofthem have only two or three colours and can only tellyou two or three different pH’s. For example, Congo redindicator paper is blue at pH 3 and red at pH 5. Litmuspaper is red at pH 5 (acid) and blue at pH 8 (alkaline)..Universal indicator papers are more useful because theyhave many colours and can tell you many different pH’s.There are several kinds of universal indicator test paper,and you may be given a paper which changes colour in adifferent way from the one we have just described. Thecover of the book in which these papers come is usuallyprinted with several differently coloured squares. Oneach square is written ‘he pH that that colour shows.Universal indicator test paper is easy to use. Put one endof a piece of paper into the solution you want to test andmatch (compare) its colour with one of the colouredsquares on the cover of the paper.The use of universal indicator test paper for measuringthe pH of the stools is described in Section 10. I I. Itsuse for measuring the pH of the gastric juice is describedin Section 11.9. It can also be used for testing the urine.1.9 Cells (FIGURE l-3)Houses are often built with bricks, and a very big housecan be built with quite small bricks. In the same way thebodies of all except the smallest living things (microorganisms-seeSection 1.11) are made of millions ofvery small cells. Cells are so small that they can only beseen with a special machine called a microscope (seeChapter Six). There are many different kinds of cells, andthey are joined together in different ways to make thetissues of the body. Skin cells are joined together to makeskin tissue. Liver cells are joined together to make livertissue. (The word tissue is also used to mean a thin pieceof paper. Lens tissue is a special thin paper used forcleaning microscope lenses.) A piece of liver is livertissue. The whole liver is called an organ. A whole eye isan organ, so is a whole heart or stomach. In this waycells make tissues, tissues make organs, and organs makethe body. This is shown in Pictures A, B, and C.Cells have a very thin outer coat called the cellmembrane. Inside the cell membrane is something calledthe cytoplasm. The cytoplasm is a mixture of manythings, especially proteins in water, and contains manysmall particles. In the middle of the cell there is a largeball or bag called the nucleus which is usually round oregg-shaped. When we talk about more than one nucleuswe say nuclei-for example, one nucleus or two nuclei.Sometimes we use the word nuclear, which meansbelonging to the nucleus. The nucleus contains a substancewhich is easily coloured (stained) and which iscalled the nuclear chromatin. Chromatin means colduredmaterial (chrom = colour). In some cells the chromatinis spread more or less evenly all through the nucleus, butin the nuclei of some protozoa it is gathered into largerparticles or lumps called karyosomes (Picture 0).Around the nucleus and between it and the cytoplasm isanother thin coat alled the nuclear membrane. If youthink of a cell as a 1 ac or bag (the cell membrane) full ofcytoplasm, then the nucleus is a smaller bag inside thecytoplasm. The parts of a cell are shown in Picture A.All cells are made from other cells. Cells divide(become two) in various ways. The usual way is for thenucleus to divide first to make two nuclei. After this thecytoplasm divides fp make two daughter cells with onenucleus in each of them, Pictures H to N show how thishappens. Most of the cells in the bodies of animals andman stay close together where they divide. In this waythere come to be more cells in a tissue, which is howtissues, organs, and people grow. The amoeba in Picture0 and the bacteria in Pictures P to T are made of onlyone cell. When one celled micro-organisms divide, thedaughter cells usually separate from one another and oneamoeba becomes two daughter amoebae. Similarly, whena bacterial cell divides two daughter bacteria are formed.There are many different kinds of cells. Picture Dshows a cell called a lymphocyte. A lymphocyte is one ofthe white cells in the blood. Picture E is an epithelial cellfrom the inside of the bladder. Picture F is a red bloodcell or erythrocyte (erythro = red, cyte = cell). A lymphocyteis round like a ball, and the kind of epithelial cellin Picture E is thin and flat. But a red blood cell has aspecial shape. It is like a ball which has been pressedtogether in the middle. Picture F shows the red cell fromthe top, and the picture underneath it shows you what itwould look like if it were cut in half along the dotted lineX-Y. A red cell is thin in the middle and thick at theedges. Red cells are unlike other cells because they havelost their nuclei. A red ccl1 is about 7+ pm across (seeSection 6.1). Red cells are red because they are filledwith a very important red substance containing ironcalled haemoglobin.Picture G shows the epithelial cells which come fromthe inside of the trachea. The trachea i ‘he tube in ournecks which takes air to our lungs. You will see that theepithelial cells from inside the trachea are fixed closelytogether, side by side. These cells form a membrane orepithelium all over the inside of the trachea. There areother kinds of epithelium inside the bowel (gut, intestine)and the urinary tract (the organs concerned with urine).The word cell can be used in several different ways.Besides its use to mean a living cell, a cell can also be aspecial glass box for putting liquids in, part of an electricbattery, part of a counting chamber, or something whichmakes electricity when light falls on it.1.10 Proteins and enzymesCells are mostly made of many different kinds of’substancescalled proteins. The proteins in our cells are

! ...;c>:1 1 lniroductionCELLS FORM TISSUES, TISSUES FORM ORGANSTHISIS A CELL\ nuclearmembraneCELLS JOIN TOGETHER TOFORM TISSUESTISSUES OF VARIOUS KINDSARE JOINED TOGETHER TOFORMORGANSSOME CELLSFROM OUR BODIESLYMPHOCYTEPlate 6cell membraneEPITHELIAL CELL(from the bladder)Plate 56RED CELLPlate 1-...,,THE TRACHEA-CELLSDIVIDINGone cellha IS divided ..the cytoplasm ’has divided.SOME MICRO-ORGANISMSEntamoeba histolytica &ytop’asmcoccusbacillusP @ Q Plate 103membraneENTAMOEBA HISTOLYTICAprotozoa1 trophozoitePlate 87\cellcocciRLsporesw a /Plate 43Plate 102SOME BACTERIAFig. l-3 Cells

Micro-organisms 1 1 .l 1made from the protein foods we eat, such as beans orfish. These proteins help to form the cell membranes andthe nucleus. They are also some of the many importantthings in the water of the cytoplasm. Proteins also formthe enzymes inside the cell which make the cell work.Enzymes are special proteins for making the things thatcells need. If you like, you can think of enzymes as beingthe machines of the cell which make the cell alive. InSection 7.41 you will read about the enzyme callednrease (all enzymes end with the word ‘-a&-forexample, lactase, amylase). Urease turns a substancecalled urea into other substances called ammonia, carbondioxide, and water. It is only one of the many thousandsof enzymes that cells have with which to make the substancesthat they need to live and grow.Most of the protein of the body is in its cells, but someproteins are outside the cells and are dissolved in theblood plasma (see Section 1.17). These are the plasmaproteins. There is no protein in normal urine, but, whenthe kidneys are diseased, the plasma goes into the urine.In Section 8.3 you will learn how to test the urine forprotein.1.11 Micro-organismsIn the world around us there are many other living thingsbesides men, women, and children. Living things arecalled organisms. Cows and dogs and trees and grass areorganisms, so are birds and snakes. Some of these organismsare very big, like elephants or big trees, and some ofthem are very small, like small insects. But, as well as theliving things we can see, there are many more livingthings which are so small that we cannot see them. Thesevery small living things are called micro-organisms(micro means small). They are so small that they canonly be seen with a microscope. The smallest microorganismsare made of one cell only. Micro-organismsare also called germs or microbes. A micro-organismcan grow and divide into two micro-organisms in asshort a time as twenty minutes. If it has the right food,one micro-organism can grow into millions of microorganismsin a few hours. Most micro-organisms live inthe soil or in water where they do no harm. Many microorganismsare useful and some help the soil. There aremicro-organisms almost everywhere. They are on thisbook, on your hands, in your nose, on tables, on floors,and on the equipment in your laboratory. There are alsomicro-organisms in the air. Most micro-organisms areharmless.1.12 Parasites, commensals, and infectionMany organisms live on or inside man and animals.Those which cause no harm are called commensals.Those which cause harm are called parasites. One of thereasons why parasites are harmful is that one parasitemay grow into millions. When someone has many harm-ful parasites inside him, he becomes sick and may die.When a person has a parasite of a certain kind livinginside him he is said to be infected by that parasite, andto be the host for it. People can be infected by dangerousmicro-organisms, and so can things such as loops (seeSection 3. lo), test tubes, and specimen containers. If aloop or specimen container has micro-organisms on it,we say that it too is infected.Each parasite has its own way of getting into the body.Some go through the skin, some get into food, and someuse other ways. Parasites can spread from one perscn toanother. When this happens, one person is said to infectanother. For example, when someone is sick with thedisease called tuberculosis, he is sick because a parasiticmicro-organism called Mycobacterium tuberculosis isgrowing inside him and infecting him. Some patientswith tuberculosis of the lungs cough sputum containingMycobacterium tuberculosis out of their lungs into theair. Sputum is the thick white or yellow substance thatpeople cough up. A healthy person may breathe in theMycobacterium tuberculosis from the little drops ofinfected sputum in the air and become infected. He mightbecome sick and die.Many of the patients coming to a health centre orhospital are diseased because there are parasites growinginside them. Most of these patients will have beeninfected by another person. A few will have got theirparasites from animals. Diseases caused by parasiteswhich can be caught from other people or animals arecalled infectious diseases. These include leprosy, tubcrculosis,ancylostomiasis (hookworm infection), schistosomiasis(bilharzia), malaria, trypanosomiasis(sleeping sickness), and onchocerciasis (river blindness).You will see that many infectious diseases end with theword ‘Gasis’. If you see a word ending in this way, youcan be sure it is an infectious disease. Many diseases arenot infectious, sickle-cell anaemia for example.Many of the methods in this book are for findingparasites in infected people. If we can find which parasitea patient has, we know what disease he is suffering from(why he is ill), and we can give him the right treatment. Ifa patient is given the right treatment, he will probably getwell.Many parasites and the infectious diseases they causeare common in almost all the warmer countries of theworld. Some of these diseases which are seen almosteverywhere are tuberculosis, leprosy, hookworm infection,Ascaris infection, gonorrhoea, and malaria. Otherdiseases are only seen in a few countries. For example,there is trypanosomiasis in Zambia but not in India. Askwhat parasites and what diseases are found in your countryand learn all you can about them. Don’t waste timelearning about diseases you will never see.1 .I 3 How organisms are namedYou may have asked yourself why we write the names ofmicro-organisms like this--Mycobacterium tuberculosis,

,:”‘”@!y, ..( r“--;-.i ,..;. ,/,., ,’ .,F.7;,I ’(.1 1 k&rod&&nEntamoeba histo&tica, or Borrelia duttoni. We do thisbecause all living things are named in this way. Forexample, man is Homo sapiens, the house fly is Muscadokestica, the maize plant is Zea mais. The first word ineach name is the tribe or genus of the organism. Whenthere is more than one genus, we say genera. The genusalways starts with a capital (big) letter. The second nameis the name for that special organism; we call it thespecies of that organism. The name of the species alwaysstarts with a small letter. In printing both names arealways written in special italic writing like this-italic.In typing or handwriting both names are underlined. Youwili meet two species of the genus or tribeMycobacterium. You will meet one species calledMJwobacterium tuberculosis, which causes tuberculosis,and another species, Mycobacterium leprae, whichcauses leprosy. The word for the genus is often shortened.Thus M.vcobacterium leprae is often written Myco.leprae, and Entamoeba histolytica is written E.histolytica. There are special shortenings for each genus,and we must not make our own.1.14 The different kinds of parasitesWe shall describe these kinds of parasite:Nematodes (roundworms) Usually bigCestodes (tapeworms) enough toTrematodes (flatworms) > seeFungi-one or more cellsProtozoa-one cell Can only beBacterEa-one cell seen with aViruses-less than one cell > microscopeViruses are the smallest kind of micro-organism. Virusesare very much smaller than a cell and can onlygrow inside the&ells of other organisms. A11 viruses aretherefore parasites. Viruses are difficult to study, and wecannot study them with the equipment in our laboratory.Viruses cause measles, poliomyelitis (polio), chickenpox,smallpox, and many other diseases.Bacteria are larger than viruses, Each bacterium isone complete cell, but it has no separate nucleus. Thenucleus of a bacterium is mixed up with its cytoplasm. Around bacterium like a ball is a coccus (Picture P,FIGURE l-3). When there is more than one coccus, wesay cocci (Picture R). A coccus usually measu&s about1 Ftm. A long rod-shaped bacterium is called a bacillus(Picture Q). When there is more than one bacillus, wesay bacilli (Picture S). Some bacteria are long, thin, andcurved like a snake. Borrelia duttoni shown in Picture Tis a bacterium of this kind. Some bacteria have’strongseeds or spores (Picture S) which are hard to kill withheat or chemicals. Tuberculosis, gonorrhoea, and manyother diseases are caused by bacteria.Protozoa are larger than bacteria and measure aboutlo-20 ktm. They are made of one cell which has a nuc-leus. We say protozoon when there is only one organismand protozoa when there is more than one. An amoeba isa protozoon which moves slowly by putting out feet orpseudopodia. An amoeba called Entamoeba histolyticahas been drawn in Picture 0. You will see the cell membrane,the cytoplasm, and the nucleus. Many protozoacan live in a larger, active moving form called a trophozoite,as well as in a smaller, ‘sleeping’, still form called acyst. Trophozoites are easily killed by dryness, sunlight,and chemicals, but cysts can often live through thesethings, and so infect new hosts. The amoeba drawn inPicture 0, FIGURE l-3, is a trophozoite. You will seecysts in Pictures E and K, FIGURE 10-S. Some protozoamove very fast by waving ‘hairs’ or flagella which stickout of the cell. Protozoa of this kind are called flagellates(see Se-+; ‘I 10.8). Organisms which move on their ownare .’ be motile. Many protozoa and bacteria aremotile. &&ozoa cause malaria, trypanosomiasis (sleepingsickness), and amoebiasis.Fungi are very simple plants. Most fungi are singlecelled (one celled) micro-organisms, but some fungi containmany cells and are large enough to see. Single celledfungi are about as wide as protozoa (about 5 jlrn), butthey may be long and thin. Their cells have nuclei andoften branch or divide into two. Unlike the protozoa,fungi are never motile; we say they are non-motile. Fungicause many skin diseases, such as ringworm.Most worms or helminths are large enough to be seeneasily. Many live in the gut (intestines, ‘stomach’) and layeggs or ova which we look for in the stool. If we findova in the stool, we know the patient must have theparent worm in his gut. Worms cause schistosomiasis(bilh@a) and ancylostomiasis (hookworm infection).1.15 Putrefaction or rottingIf meat. milk, blood, or urine are left in a warm place,they soon start to smell and go bad. They are said toputrefy or rot. This is because micro-organisms, especiallybacteria, grow in the meat, the milk, or the bloodand destroy or spoil them. Some reagents, such as antiseraor bovine albumen (see Sections 12.3 and 12.6) alsoputrefy and spoil if they are kept warm. One way to stopthings putrefying is to keep them cold in a refrigerator.Micro-organisms cannot grow when it is cold, or theyonly grow very slowly. Micro-organisms seldom growwell enough in ordinary chemicals and reagents to spoilthem, because most reagents do not contain the rightthirigs for micro-organisms to eat. Micro-organisms will,however, grow in a solution like sodium citrate. Citratesolutions should therefore be kept in a refrigerator.1.16 StainsBecause the cytoplasm of cells’ is a mixture of things inwater, living cells are clear and watery and are often

Stains 1 1.16difficult to see. We usually therefore colour cells withstains. We say we stain them. A stain is a coloured liquidlike ink. In Picture D, FIGURE l-3, the nucleus of thelympocyte has been stained and looks black; so also havethe bacteria. But the epithelial cell in Picture E is drawnunstained. Several stains are used in the methods describedhere. These stains are Gram’s stain, Leishman’sstain, crystal violet, and carbol fuchsin.1.17 Serum and plasma (FIGURE l-4)When blood comes from the body it is a thick red liquid.Picture B, FIGURE l-4, shows blood coming from a fingerprick. The finger has been pricked with a glass chip-seeSection 4.7. If blood is put into an empty tube, as inPicture A, it soon goes solid as in Picture C. It is said toclot or coagulate. If the blood clot is left for some hours,you will see that a clear yellow liquid comes out of theclot and that the clot retracts (gets smaller) as in PictureD. This clear liquid that comes from clotted blood iscalled sernm..We can easily stop blood clotting by adding a specialchemical to it; Chemicals for stopping blood clotting arecalled anti-coagulants. The anti-coagulants used in thisbook are sequestrene (Section 4.6), sodium citrate(Section 7.39), and heparin (Section 7.2). Sequestrenehas many names. It is also called potassium EDTA,potassium ethylene-d&nine-tetra-acetic acid, and sequestricacid potassium salt-don’t learn these names, butremember that you may see any one of them on a bottle.You must have the potassium salt; plain sequestric acidwill not work. If a very little sequestrene is put in thebottom of the tube, as in Picture E, FIGURE 1-4, andblood mixed with it, as in Picture F, the blood will stayliquid and will not clot. It can easily be poured intoanother tube as in Pictures G and H. If the blood is leftuntil the following morning, it will remain liquid. The redcells will fall to the bottom of the tube. The slightlycloudy yellow liquid on top is called plasma. On the topof the red cells is a thin dirty white layer. Most of thewhite cells are in this layer: it is called the white celllayer. Some people call it the buffy coat. Althoughplasma and serum are both yellow liquids and both comefrom blood, they are not the same. Serum comes out of ablood clot. Plasma is the liquid part of blood which hasbeen prevented from clotting.fingerPLAINBLOODglass chip 10 MINUTES LATERNEXTDAYempty ___testtubeSERUMthe blood hasbecome solidthe clothas retractedBLOOD WITH AN ANTICOAGULANT /‘6ENEXTDAYa littleanticoagulant-(sequestrene)the blood isstill liquidand can bepoured intoanother tubeFig. l-4 Serum and plasmaPLASMAthis is thewhite celllayerred cellssedimented

1 1 IntroductionIn most methods for blood in this book unclottedspecimens in sequestrene are used. But in blood grouping,for example, clotted blood is always used. In somemethods, such as the serum urea, either clotted or unclottedblood can be used. Because it is important to haveeither a clotted or an unclotted specimen, whichever themethod says, blood must be taken into the right kind ofbottle or tube. Laboratories therefore give the wardsplain empty bottles for clotted blood and bottles withsequestrene in them for unclotted blood. Making thesespecimen bottles or tubes is described in Section 4.6.1 .I 8 Isotonic solutioimAs you have read, the cytoplasm inside the cell memraneis a mixture of many different things in water. Oneof these things is salt. In a living healthy cell there isalways just the right amount of salt in the cytoplasm.Some cells, especially some bacterial cells, do not mindhow much salt there is outside the cell membrane in thesolution around them. These cells will live quite wellwhen there is a lot of salt in the water around them orwhen there is very little. If there is much salt outsidethem, they can stop too much getting in through the cellmembrane. If there is little salt outside them, they canstop too much getting out.But the red cell is different. The red cell can only livein a solution in which there is just the right amount ofsalt. If thtre is too much salt in the solution around a redcell the water inside it goes out through the cell membrane.The cell therefore gets smaller because it has lostwater and shrivels up (folds up). Shrivelled up red cellsare said to be crenated-look at Picture C, Figure 7-15.If there is too little salt in the solution around a red cell,the water outside comes in through the cell membrane.Because water has come in the red cell swells (gets bigger),it becomes round and then bursts or lyses or haemolyses(breaks open). If red cells are going to stay theirright shape, there must be the same amount of salt outsidethem as there is inside them. We call a saline (salty)solution of just this right strength isotonic saline(iso = equal, tonic = strength). An isotonic solution hasthe same salt concentration as the cell cytoplasm.Weaker saline solutions, which make the red cell swellup and burst are called hypotonic (hypo = less). Strongersolutions which make the red cell shrivel up and crenateare called hypertonic (hyper = more). A hypertonicsaline solution will make red cells crenate. An isotonicsolution keeps red cells healthy. A hypotonic saline solutionwill make red cells swell up and burst. An isotonicsaline solution for red cells contains O-,3$ of sodiumchloride (common salt). That is, it contains 0.85 g of saltin 100 ml of solution (see Section 3.40). This isotonicsolution is sometimes called physiological saline or normalsaline. Often, as in this book, it is just called ‘saline’.The saline you read about therefore means 0.85% ofsodium chloride in water. It is mostly used for washingred cells for blood grouping.Don’t muddle up normal saline, which is for washingred cells and which is only salt and water, and form01saline, which is salt, formalin, and water. Form01 salineis for fixing or preserving tissues (stopping them putrefyingor rotting).As you have read, when red cells break open and thehaemoglobin inside them comes out, they are said to lyseor haemolyse. Many substances will make red cells haemolysebesides water or a hypotonic solution. Soap, or adetergent such as ‘Teepol’, will make blood haemolyse.For some methods, such as the serum urea, it is importantthat blood does not haemolyse before it is examined.When we measure the haemoglobin we want to lyse redcells, so that the haemoglobin comes out of them. We dothis by putting a little blood into a much larger volume ofa very dilute alkaline solution made of sodium carbonateor ammonia.1 .I 9 Disinfectants and antisepticsWhen patients have micro-organisms inside them that’are causing disease, we kill these micro-organisms bygiving the patient drugs (medicines). These drugs arecarefully chosen so that they kill the micro-organismsbut not the patient. For example, if the patient is infectedwith Mycobacterium tuberculosis he may be treated withdrugs called streptomycin, PAS, thiacetazone, and INH.These drugs kill the mycobacteria inside the patient’sbody. Often we want to kill harmful micro-organismsoutside the body. For example, we must killMycobacterium tuberculosis in sputum bottles that aregoing to be washed. We want to kill any parasitic microorganismsthat we have found in a patient’s sputum. If wedo not do this we might ourselves be infected with aparasitic micro-organism and suffer from a disease. Wecannot use drugs to kill these micro-organisms, becausedrugs are too expensive. Also, they only kill some microorganisms.Instead we use chemical solutions calleddisinfectants. To disinfect something means to kill theharmful micro-organisms in it, or to remove the infectionfrom it.Disinfectants kill most organisms. They will kill ustoo, if we drank them. So they cannot be used as medicines.Most disinfectants would harm the skin if theytouched it. There are many disinfectants. A common oneis a brown oily liquid called lysol. Keep a jar of lysol onyour bench and put into it anything you have finishedwith and which might be infected and might thereforebe dangerous. Keep a bucket of lysol under your benchand put infected specimen bottles into it. Make thelysol solution for the bucket by ddding half a cupfulof pure lysol to a bucket about three-quarters full ofwater.Some people like to keep a pressure cooker on theirbench with a little water in it. They put everything whichis infected into it, such as sputum pots. When the cookeris full it is quickly heated on a paraffin pressure stove.

Stklization 1 1.20This kills all the harmful organisms, and the things insidethe cooker can then be safely washed.Antiseptics are half-way between drugs and disinfectants.Antiseptics kill micro-organisms and may kill youif you drink them. But antiseptics do not harm the skin,so they can be used quite safely to kill micro-organismson the skin. Antiseptics are used to kill micro-organismson the skin before a surgical operation. This stops themgetting into the patient’s wound. Spirit, iodine, and.cetrimideare antiseptics.1.20 S wilizationThere are other ways of killing micro-organisms. Oneway is to heat them so hot that they die. By heatingsomething in the right way we can kill all the microorganismsin it, including all the bacterial spores. Allmicro-organisms can be killed by heat, not only most ofthem as in disinfection. When we kill all the microorganismsin or on something we say we sterilize it.Something in which all the micro-organisms have beenkilled is said to be sterile. In our laboratory the thing thatwe sterilize most often is a wire loop (look at Picture C,FIGURE 3-2). A loop is usually sterilized in the flame of aBunsen burner or a spirit lamp. The end of a Pasteurpipette or the surface of a slide can also be sterilized byheat in a flame. When we sterilize something by putting itthrough a flame, we say we are flaming it. When a loophas been flamed all the micro-organisms on it will havebeen killed and it will be sterile.Another way of sterilizing things is to put them intoboiling water. All hospital wards have special boilingwater sterilizers for sterilizing such things as bowls, scalpels(the knives surgeons use), and scissors. Boilingwater is not a good way of sterilizing things because it isnot hot enough. But we cannot make a pan of boilingwater any hotter. If we heat the water more it only boilsaway faster and does not get any hotter. The boilingwater turns into steam and goes into the air. If water is tobe made hotter than boiling water the steam must bestopped from getting away. To stop the steam gettingaway, the water must be heated ii: a special strong panwith a strong tight cover (lid). The steam tries very hardto force (push) its way out and would burst a weak panopen or push the lid off. When the steam pushes stronglyin this way but is stopped from getting out, we say thesteam is under pressure.Pressures are measured in pounds per square inchwhich is often written lb. sq. in. Pounds is shortened tolb., square to sq., and inches to in. In the next section, forexample, you will read about a pressure of 15 lb. persq. in. This means that on every part of the inside of thepan and its lid one inch long and one inch wide (onesquare inch) the steam is pressing just as if it were aweight of 15 lb. When something is heated in puresteam at 15 lb. for 15 minutes, all the micro-organismsin it will usually be killed, and it will be sterile. ‘ 15 lb. for15 minutes’ is an easy sterilizing pressure and time toremember.The harmful micro-organisms in specimens of stool,sputum, etc., will be killed by steam at a pressure of15 lb. for 5 minutes. So we need only autoclave for 5minutes to disinfect specimen containers and make themsafe to wash. They will not be sterile (some microorganismswill be left, including most bacterial spores),but they will be safe to wash (the harmful microorganismswill have been killed).A pan for sterilizing with steam under pressure iscalled an autoclave. In a boiling water sterilizer thethings to be sterilized are kept under the water. In anautoclave the things to be sterilized are kept in the hotsteam above the water. Every hospital has a big autoclavefor killing the micro-organisms, including thespores, in the towels and dressings (bandages, gauze, etc.)that are used for doing surgical operations. Spores arevery dangerous in surgical dressings because one kind ofbacterial spore can cause a disease called tetanus. Largelaboratories also have big autoclaves. The best kind ofsmall autoclave for a small laboratory is called a pressurecooker. Don’t muddle up a pressure cooker which is asmall autoclave and a pressure stove (ML 7 1) which is astove using paraffin or kerosene under pressure. A par-&in pressure stove is also called a ‘Primus stove’. Pressurecookers are made for cooking food in kitchens,because cooking, like sterilizing, goes faster in steamunder pressure.1.21 The pressure cooker (FIGURE l-5)The pressure cooker in the main equipment list is calledthe Prestige ‘Hi-Dome’ pressure cooker. A small bookcomes with the cooker which tells you how to use it forcooking food. This section tells you how to use thecooker for sterilizing equipment in a laboratory.In Picture A you will see that there are two main partsto the cooker: a body (the panj and a cover (the lid). Thepan and the cover both have long handles. The cover fitstightly on to the pan, and between them there is a rubberring called the gasket (Pictures A and Q). On top of thelid there is a 3-way control valve (a valve is a tap) and asafety plug (a plug is a cork or stopper).Footnote. When this book went to be printed the Prestige ‘Hi-dome’pressure cooker was still being made with weights that measuredpressure in pounds per square inch, and not in kilograms per squarecentimetre. This is why pounds per square inch have been usedthroughout this book. If you have a pressure cooker that measurespressure in kilograms per square centimetre, you will need to know thefollowing:I5 pounds per square inch is nearly equal to one kilogram persquare centimetre (kg/cm’).IO pounds per square inch is nearly equal to 0.7 kilograms persquare centimetre.5 pounds per square inch is nearly equal to 0.3 kilograms persquare centimetre.

1 1 IntroductionA THIS 1s THE PRESSURE COOKER 6CUT IN HALF \ . THREE-WAY CI ONTROLCVALVE, middle weighthandle of coverweightsyringes inside being’ ’ ’sterilized/trivetor shelfwhen all three weights are’ beingused there has to be a highpressure of steam inside thecooker before it can get outG THE PRESSURE COOKER HAS BEENCUTIN HALF TO SHOW WHATIT LOOKS LIKE INSIDE-..---3wavM-L I IlKtt VVtlbll IShi.Anmm / controlSCREWED TOGETHER,,“,;“‘“\--Ah---valve(this is the sameas in Dir+,,m t-lLP“’ ’ ‘rru’r’ “’ .-top handle/-~-~+. -Xx+2 ‘._. 1‘i--.. I safetyH SAFETY PLUG IN(as in normal use)1 SAFETY PLUG OUT(steam is getting out)EINSIDE AND MIDWEIGHT SCREV aTOGETHERsafety IIlocking ”14J HOW TO PUT THE LID ONINSIDE WEIGHT(this is the samebaseu @ arrowsas Picture ‘) HOW TO FIT THE HANDLES TO YOUR PRESTIGE PRESSURE COOKERInsert the longer of the two handles into theK m bracket on the lid.-Insert screw-end rod into handle and screw up tightly.REPLACEMENTSNozrepeat the process by inserting the smaller Lhandle in the bracket on the body, as shown indiagram (B) and secure firmly in position in thesame way as (A)Q gasket, three-way/ control valveN upper handleP fronthandleaR safety plugFig. l-5 The pressure cooker

The pressure cooker 1 1.21The 3-way control valve controls (adjusts) the pressureof the steam in the cooker. It is called 3-way becauseit can control the steam at three different pressures. It ismade of three weights which screw together. There is aninside weight (Pictilres B and F) and two weights likerings which screw on to it. Picture E shows the insideand middle weight screwed together. Pictures C and Dshow the inside, the middle, and the outside weightscrewed together. A rod on the inside weight blocks(shuts) a small hole in the lid of the cooker. This hole iscalled the vent. The steam has to lift the rod and theweights before it can get out of the vent. If the weightsare heavy (as in Picture C) there has to, be a high pressureof steam in the cooker before the s’team can lift therod and the weights and get out of the vent. By using thethree weights of the 3-way control valve we can controlthe pressure of the steam inside the cooker at 5, 10, or15 lb. per sq. in. In Picture B only the inside weight(Picture F) is being used, and the steam can get out of thecooker when it is only at a pressure of 5 lb. per sq. in.When the middle weight is screwed on to the insideweight the steam can get out when it is at 10 lb. per sq.in. In Pictures C and D all three weights are screwedtogether, and the steam can only get out when it has gotto a pressure of 15 lb. per sq. in. Whenever you sterilizethings in a cooker use all three weights togetherthatis, at 15 lb. per sq. in. and keep this presure for 15minutes. It is possible to disinfect things by killing themore harmful micro-organisms and so making themsafe to wash, by keeping a pressure of 15 lb. for only5 minutes.If tLe vent becomes blocked (shut off so that steamcannot get out), the pressure of steam in the cookermight get so high that the cooker would burst (explode orsuddenly break open). This would spoil the cooker andmight hurt you. A safety plug (Pictures H, I, and R) hastherefore been put on the cooker to let out the steamwhen the pressure becomes too high. In this way a safetyplug stops the cooker bursting. The safety plug is a pieceof round black rubber with a hole in it. A speciallyshaped metal rod sticks into this hole. When the cookeris being used this rod sticks into the hole, as in Picture H,and keeps the steam in the cooker. But, if the steampressure rises too high (that is, much higher than 15 lb.per sq. in.) the rod of the safety plug comes out (as inPicture I) and lets out the steam. When the steam hascome out the rod can easily be pushed back again. Thecooker will. not work with the rod sticking out becausethe steam will get out too easily. This is why Picture I iscrossed out.Three little baskets, the separators, come with thecooker, and are useful in the laboratory. There is also ametal circle (disc) with holes in it. This is the trivet (atrivet is a shelf for holding things while they cook), Youwill see that the trivet has a rim or edge on one side only.Always use the trivet with the edge down. In this way thetrivet will keep the things you are sterilizing from beingwet by the water.Steam is much better than hot air for killing micro-organisms. When you start sterilizing it is therefore veryimportant to let the steam from the boiling water blowaway all the air. Wait until the steam is coming out of thevent in a steadyflow before you put on the weights. Whenthe steam is coming out in a steady flow it will haveblown sway the air and then there will be pure steaminside the cooker. This pure hot steam will kill the microorganisms.When the air has been blown away you canput on the weights and let the pressure in the cooker riseto 15 lb. per square inch. A little steam will escape whilethe pressure is rising. When 15 lb. pressure has beenreached steam will again start escaping fast. You canthen turn the heat down a little and start timing 15minutes.You now know enough about the cooker to be able touse it. Perhaps you want to sterilize some syringes, asdescribed in Section 4.9. Before you start you mustremember one thing. This is that THERE MUSTALWAYS BE SOME WATER AT THE BOTTOM OFTHE COOKER. This water makes the steam for sterilizing.If there is no water the equipment in the sterilizerwill burn and the cooker will be spoiled.METHODUSING THE PRESTIGE ‘Hi-DOME’ PRESSURE COOKERUnpack the cooker. Screw the handles on to thepan and the cover, as shown in Pictures K and L.Put the trivet in the cooker with its rim (edge)downwards. Put two cupfuls of water in the cooker.The top of the trivet should be just dry.Put the syringes, or whatever else is to be sterilized,into the cooker. Put them in an empty tinwithout a lid, so that they keep as dry as possible.Loosen the lids of any bottles so that air can getout and steam can get in. Take the plungers out ofthe syringes as in Picture F, Figure 4-3. If you don’tdo this the barrels of the syringes may break.Put the cover on the pan. Put it so that the arrowyou will see on the edge of the cover is in line withthe arrow you wiil see on the handle of the pan, asshown in Picture J. Move the handle of the cover tothe left until both handles are together as shown inPicture G. The cooker is now closed.Make sure you have removed the weights of thepressure control valve.Put the cooker on a paraffin pressure stove andheat it strongly. In a few minutes steqm will startcoming out of the control valve. Wait uritil steam iscoming out in a steady flow and has had time toblow away all the air from inside the cooker.Screw together all three weights of the 3-way controlvalve (as in Picture D) so that it works at 15 lb.per sq. in. Put the weights of the 3-way control valveon the vent and push them down. Steam will stop comingout, except perhaps for a very slight hiss (a hissis the noise steam makes when it is coming out of ahole). Leave the cooker for 2 or 3 minutes with the

1 1 Introductionheat still high. During this time the pressure will riseto 15 lb. per sq. in.As soon 8s the steam comes out fast and starts tomake 8 really LOUD hissing noise turn the flame lowby opening the tap on the stove 8 little. Start timing15 minutes with your watch. Adjust the heat so thatthere is a SLIGHT hissing noise throughout the 15minutes. *-At the end of 15 minutes take the cooker off thestove. Leave it on the bench to cool. You will knowwhen it is cool because no steam will come outwhen you lift the weights. Always let the cooker coolslowly when you are sterilizing liquids in botdes. Ifyou want to cool the cooker fast, hold it under a tapso that water runs over it, or cover it with 8 verywet cloth. This will turn the steam inside the cookerback to water again. Lii up the weights of the3-way valve a liile after about half a minute. Thiswill tell you if there is any steam still left. If there is,put the cooker back in the water for a few more minutes.When there is no steam left, take off the weightsof the $-way control valve. Next take off the coverby moving the top cover handle to the right.All the micro-organisms inside the cooker will havebeen killed. and the syringes will be sterile.Equipment inside a tin will probably sterilize betterif you lay the tin on its side to let out the air. Neversterilize anything in 8 tin or bottle with the lid on.1.22 Using sterile equipment. Aseptic precautionsIn larger laboratories autoclaves and pressure cookersare used for sterilizing many different kinds of equipment.But with the methods in this book the pressurecooker is only used for sterilizing syringes andneedles (Picture G, FIGURE 4-3), ‘needles and rubbertubes’ (Picture E, FIGURE 4-3), bottles of sodiumcitrate solution for the Westergren ESR, and bufferfor gastric washings. The- cooker is also used forsterilizing Pasteur pipettes (Section 3.9) and specimenbottles (Section 4.10), and for making infected specimencontainers, etc., safe to wash up. A pressure cookercan also be used for sterilizhg syringes and needles foruse in a clinic or health centre. If you are going to use apressure cooker to sterilize plastic syringes, sterilize onlyone syringe to begin with. Many kinds of plastic arespoilt in a pressure cooker, but things made of strongplastics, such as nylon or polypropylene. will not spoil.SOME WPOFITANT THINGS TO REMEMBERUse ‘your cooker either for sterilizing clean thingssuch as syringes, or for disinfecting dirty things suchsxputum containers. Don’t try to sterilize and disinfecttit the same time.Keep a spare gasket and a spare safety plug. Aswith all rubber equipment, keep them in the dark, orthey may spoil. There are other spare parts you canget. These spares or replacements are shown at thebottom of Figure 1-5.NEVER LET THE COOKER BOIL DRY. Unless alot of steam is lost, two cupfuls of water will beenough. Don’t let the cooker lose too much steam, orit will boil dry and spoil.Start hearing with the 3-way control valve OFF.Don’t put it on until there is a steady flow of steam.This is very important. The flow of steam blowsaway the air so that things can be sterilized in puresteam. ‘15 lb. for 15 mins.’ will only sterilize if allthe air has gone and pure steam is left.Don’t start timing 15 minutes until you have putthe 3-way control valve on.Make sure that both handles are together andthe rod of the safety plug is in before you start heating.Don’t try to open the cooker after heating untilyou have let down the pressure of steam by coolingit in water.Never use the cooker more than half full of liquidor two-thirds hrll of solids.Keep the vent clean.‘Asepticprecautions’In the laboratory this means doing something withsterile equipment while taking great care to keep theimportant part of it sterile. Aseptic means sterile orwithout infection. For example, in Section 4.10 youare told to separate serum from clotted blood aseptically.You are asked to put it into a sterile bottle andsend it to a central laboratory. Because you havetouched the outside of the bottle there will be microorganismson the outside. These micro-organismsmust not get inside the bottle into the serum. In thewards using aseptic precautions means doing something,like dressing a wound, without getting microorganismsinto the wound. If micro-organisms getinto the wound, it may become infected or septic.Aseptic precautions are especially important in lumbarpuncture which is described in Section 9.5,The first thing to remember is that there are microorganismseueryyjhere, except where they have beenkil!ed by sterilization. There are micro-organisms onyour hands, on the bench, and on every piece ofequipment which has not been sterilized. When youtouch something sterile with your fingers the partwhich you touch is 110 longer sterile. This part is nowcovered with micro-organisms from your *fingers. Thismeans that when you use a sterile bottle, Pasteurpipette, or syringe, you must touch one part only andleave the other parts sterile. You may only touch theoutside of a syringe. You must not touch the plunger(piston or inside part), and you must not touch thenozzle (the place where the needle goes). If you touch

Laboratory infection 1 1.23the plunger, micro-organisms will get from your fingerson to the plunger and so into the patient’s blood.You will learn what we mean by ‘aseptic precautions’from the following example. We will describehow blood can be taken from a patient and the serumseparated from it aseptically.METHODTAKING A BLOOD SPECIMEN AND SEPARATING THE SERUMASEPTICALLYWii a pressure cooker sterilize a syringe (Section4.9). some plugged Pasteur pipettes (Section 3.9). andsome bijou bottles or universal containers. The bottlesand the syringe must be dry if the serum is not going tohaemolyse (Section 1 .18).TAKING BLOOD ASEPTICALLYTie a rubber tube round the patient’s arm and choosea good vein as shown in Figure 12-7. Put a piece ofcotton wool or gauze in spirit or iodine end swab (paint)the skin where you are going to put the needle. Theiodine or spirit is being used as an antiseptic to killmicro-organisms on the skin.Carefully unwrap the syringe from the paper. Pick upthe plunger by its handle. Don’t touch the main part ofthe plunger. Put the plunger into the barrel withouttouching the nozzle (the place where the needle fits).Pick up the needle by its adapter (thick part). Put theneedle on to the syringe without touching anywhere theblood might go.Put the needle into the vein. as shown in Figure 12-7,and fill the syringe with blood. Don’t touch either thepoint of the needle or the plunger of the syringe as youdo so. Take out the needle when the syringe is full ofblood. Press over the hole that you have made in thevein with a piece of cotton wool and ask the patient tokeep pressing it for a minute or two. This will stop theblood from the vein bleeding into the tissues of the arm.Take the needle off the syringe.Loosen the cap of the bottle with your index fingerand thumb (look in Figure 3-8 if you do not know thenames of your fingers). Take hold of the cap of the bottlein the little finger of your right hand. Turn the bottleround with your left hand. This unscrews the bottle fromthe cap.Put the blood into the bottle without touching ‘anypart of the bottle with any part of the syringe.Still holding the cap in your right hand, screw thebottle back into it. By doing this the blood hes touchednothing which has not been sterilized. Nothing unsterilehas touched anywhere where the blood will go. Theoutside of the bottle end cap will be covered with microorganisms,but the blood inside will be sterile.Leave the blood until the serum has separated (Figurel-4).SEPARATING SERUM ASEPTICALLYTake a sterile plugged Pasteur pipette (Picture K,Figure 3-51 and break off its closed end. Fit a teat to it.Loosen both the cap of the bottle with the blood in it andthe cap of the empty bottle into which the serum isgoing to be put.Hold the cap of the empty bottle in your little finger asdescribed above, and unscrew the bottle from it withyour left hand.Take off the cap of the bottle with the clotted blood init.Flame the end of the Pasteur pipette and quickly let itcool. Suck up the serum from around the blood clot. Putit into the empty bottle.Flame the neck of the bottle which now containsserum and screw it back into the cap which is still heldin your right little finger.When you use a sterile Pasteur plugged pipette youhave first to flame it because only the inside is sterile.When they take blood aseptically, many people flamethe nozzle of the syringe and the needle before they putthem together. This kills any micro-organisms there maybe on the nozzle or on the needle. They also flame thenozzle before they put the blood into the bottle and theneck of the bottle before they screw on its cap. Theflame of a spirit lamp is often the easiest one to use.This is one of the few aseptic methods that are describedin this book. It is one of the few methods whereyou have to stop the ordinary and usually harmlessmicro-organisms on your fingers getting into a specimenor bottle. But, in many methods you must stop theharmful micro-organisms in a specimen getting out of aspecimen and around the laboratory. Dangerousmicro-organisms may make you ill, or possibly even killyou. We will say more about this in the next section.1.23 Laboratory infectionMany dangerous micro-organisms come into the laboratoryin specimens from patients. Mycobacterium tuberculosiscomes into the laboratory in sputum coughed upby tuberculous patients. There are also many dangerousmicro-organisms and worms in patients’ stools. Cerebrospinalfluid (CSF-see Section 9.16) may contain adangerous micro-organism called the meningococcus.All these micro-organisms and others may infect alaboratory worker. How then can you stop yourselfcatching diseases from. the specimens that vou look at?Prevent yourself being infected by following these instructionsvery carefully.METHODHOW TO STOP CATCHING DISEASES FROM INFECTEDSPECIMENSKeep all specimens in the bottles in which they cameor on the slides, wire loops, or tubes that are meant for

_~;I’.-c,,.1 1 Introductionthem. Don’t get specimens on to the floor, the bench.your hands, your clothes, or on to the outside of anyequipment that is not meant for them. If you spill 2specimen, cover it with lysol or some other disinfectant.Keep a bowl of lysol on your bench for this purpose. Onepart of Iysol in nine park of water is enough.Don’t eat, drink or smoke in a laboratory. If you put 2cigarette on the bench and then in your mouth, you willdeserve any disease that you get.Never pipette a specimen of CSF with the kind ofpipette that you put into your mouth. You may bymistake get the CSF into your mouth. Some specimensof CSF are very dangerous. Always pipette CSF with aPasteur pipette and a teatAlways wash your hands when you leave the laboratoryto go home, and especially before you eat. So, keeptowels and soap in your laboratory.Put all infected specimens, such as sputum or stools,into a bucket of disinfectant before their containers arewashed. You can, if you wish, put them straight into apressure cooker.Remember that even blood from a healthy personmay be dangerous. It may contain micro-organisms thatcause jaundice. So be just as careful with blood specimensas you are with specimens of stool or CSF. Trynot to get blood on your hands and do not spill bloodabout the laboratory.Don’t leave uncovered specimens on the bench. Fliesmay get into the specimen and carry micro-organismsfrom the specimens to food that is going to be eaten.1.24 ControlsSometimes it is difficult to know if a method is workingor if it is not working. The way to find out is to do‘control tests’ or controls. By control tests we mean testson specimens that we know are positive (a positive control)and tests on specimens we know are negative (anegative control). For example, if you are not sure ifyour sickle-cell method is working, do the sickle-cell teston a specimen you are sure is positive and on one youare sure is negative. If you get the right answer, yourmethod is working well. If you get the wrong answer,there is something wrong with your method. Controlsare only talked about for some methods in this book,such as those for sickle cells and blood grouping. Butyou may want to do controls for any method!QUESTION-S1. Give two examples each of an acid, an alkali, a salt,a buffer, and an indicator.2. Which of the following pH’s would mean thatthe solution was acid-pH3, pH10, pH7, pH8, pH6,pH4? At which of these figures would the pH beneutral?3. What are the differences between viruses, bacteria,and protozoa?4. What parts of a cell do you know? Draw a pictureof a cell.5. Describe the more important ways in which youcan prevent yourself becoming infected in a laboratory.6. Draw a pressure cooker and describe how youwould use it to sterilize some syringes.7. What is sequestrene, and why is it used?8. What are ‘aseptic precautions’? Describe onemethod, either in the laboratory or in the ward, in whichaseptic precautions are used.9. What is the difference between (a) serum andplasma, (b) an antiseptic and a disinfectant, (c) genus andspecies, (d) an organ and a tissue, (e) a coccus and abacillus.10. What is the difference between (a) a filtrate and asupernatant fluid, (b) a symptom and a sign, (c) a fungusand a helminth, (6) zero and infinity, (e) the way in whichsections and figures are numbered in this book.

2 1 Equipment andChemicals2.1 The equipment describedIn this chapter you will read about the smaller pieces ofequipment and the chemicals you will need. The balancesand the measuring instruments are described in ChapterFive and the microscope in Chapter Six. ChapterThirteen describes all this equipment more fully and isfor the storekeepers who must order it.There is a picture of almost every piece of equipmentin the figures in this chapter, and many pieces of equipmentare also shown in other parts of the book. Thecomplete set of equipment is for a small hospital laboratory.A few things will not be needed by a health centreand have been marked ‘Hospitals Only’.To help with ordering, each piece of equipment hasbeen given an ‘ML number’. ML stands for ‘MedicalLaboratory’. Because the equipment list changed as thisbook was written some numbers had to be left out andothers added. You must not therefore expect the numbersto be complete. In some figures in other chapters MLnumbers have been put beside pieces of equipment. Thiswill help you to find the equipment in the list. Besideeach piece of equipment in the figures in this chapter iswritten the number that should be ordered for supplyinga health centre. Hospitals need more of mostthings.There is an asterisk (an asterisk is a star like this *)beside the pictures of some pieces of equipment. Thisasterisk means that the equipment is only made by onemaker, and that no other equipment should be supplied.The equipment is not in alphabetical order in thefigures here, because in this chapter we have called thingsby their ordinary names and not by their cataloguenames. In Chapter Thirteen the equipment is in alphabeticalorder by its catalogue name. As used here a catalogueis a book which describes equipment. For example,the piece of equipment numbered ML 12 is called a hz ndcentrifuge in FIGURE 2- 1. It is called a ‘CENTRIFUGE,hand, . . .’ in Section 13.8.As in many other figures the equipment is not drawnto scale. Thus, although two things may be drawn thesame size in the figures in this chapter, they may not bethe same size when you see them.The more important equipment is in the main list,both in this chapter and in Chapter Thirteen. This hasbeen divided into a special list, which is equipment thathas to be bought from makers of special laboratoryequipment, and an ordinary list. The ordinary list isequipment like buckets and cups that can be boughtin ordinary shops. Sometimes you will be expectedto use other equipment and other chemicals. Thesehave been put in a list of choices in Chapter Thirteen.We shall describe the main list first and then thechoices.2.2 Special equipment in the main list (FIGURES2-1, 2-2, 2-3)ML 1, 2, 3 are the Ohaus balance and the things that gowith it. The balance is described in Section 5.5.ML 4 is the Westergren blood sedimentation tube, orESR tube. It is the long thin tube shown in Picture 10,FIGURE 7-33. These Westergren sedimentation tubes areused with the Westergren stand ML 11.ML 5 is a polythene dropping bottle. It holds about125 ml and has a top with a spout (tube). When you holdthe bottle upside down the liquid inside falls out drop bydrop. This is often very Jseful. Many of the reagents forthe methods in this book are kept in polythene droppingbottles. Sometimes, in a new bottle, you will find that thehole in the spout is blocked. If it is, make a hole in theend of the spout with a pin or needle. Use droppingbottles for these reagents: 10% barium chloride, strongcarbol fuchsin. dilute carbol fuchsin, white blood celldiluting fluid, crystal violet, Ehrlich’s reagent, Fouchet’sreagent, Lugol’s iodine, 3% salicylsulphonic acid, 20%salicylsulphonic acid, malachite green, Pandy’s reagent,ferric chloride solution, and saturated sodium acetate,etc.ML 6 is a litre (1,000 ml) polythene reagent bottle.Ordinary glass bottles can be used, but they are oftendifficult to find in health centres. These special plasticbottles have therefore been put in the list. They are forreagents that are needed in larger volumes than will gointo the dropping bottles (ML 5). Use these reagentbottles for Benedict’s solution, strong carbol fuchsin,

* this means that a piece of equipmentis made by one supplier onlyML 2 A scoop and counterweightfor the Ohaus balanceML3 Attachment weightsfor the Ohaus balancethese are the numbersteat,.:. ..-.__. 25 mlml:_:..-_’.:--.iML 4 Westergrenblood sedimentationtubesML 5 PolythenedroppingbottlesML 6 Polythene ML 7 ‘Polystop bottle’ ML B Polythenereagent bottleswash bottle1 1/L.I u-I//irubbertuber’25)_.d-___00 :oooI;!i;lML 9 Test tubebrushML 10 Bunsen burnerML 11 Westergren standscrewto fix onto’a benchML 12 l-landcentrifugemetal cap with rubber500 , ._ _:J,...ML 13 LovibondcomparatorML 14a Bijou ML 14b Universal ML 14c’Polypot’bottlecontainerFig. 2-l Special equipment in the main list-oneML 14d ‘Polytube’

Special equipment in the main list 1 2.2Ehrlich’s reagent, fontto saline, normal saline (don’tmuddle up these last two reagents-see Section 1.18),haemoglobin diluting fluid, and Pandy’s reagent. Youwill find that it is sometimes useful to keep a smallvolume of reagent for daily use in a dropping bottle,and a larger volume in reserve in a-reagent or stockbottle.ML 7. A ‘Polystop’ bottle is a lOO-ml glass bottlefitted with a polythene stopper. A stopper is a cap orcork. The stopper of a Polystop bottle fits the bottlevery tightly. A glass tube goes through the stopperand down inside the bottle. This tube is called a pipette(a pipette is a small pipe or tube). It has a little rubberbag on it called a teat. Liquid can be taken out of thebottle with the pipette and teat and dropped out of thepipette one drop at a time. You will need four Polystopbottles, one for Leishman’s stain, one for Leisbmaubuffer, one for normal saline, and one for Lugol’siodine.ML 8. This is a polythene bottle with a tube goingthrough the cap. When you squeeze the bottle (press itbetween your fingers and thumb), liquid comes outthrough the tube in a stream and not in drops as it doesin a dropping bottle. This kind of bottle is called a‘wash bottle’ or a ‘squeeze bottle’. Use wash bottles forwater, 3% acid alcohol, Benedict’s solution, diluteLeishman buffer, haemoglobm diluting fluid, and normalsaline.ML 9 is a test tube brush for cleaning test tubes. It ismade of twisted wire with stiff bristles (hairs).ML 10 is a Bunsen burner. It bums gas from a cylinder.A cylinder is a heavy steel bottle (ML 60) which canbe refilled with gas when it gets empty. Cylinders arerefilled in a factory. The Bunsen burner is joined to thecylinder by a rubber tube (ML 50). If you have no gasyou will have to use a spirit lamp (ML 39) or a paraffinpressure stove (ML 71).ML 11 is a stand for the Westergren sedimentationtubes described under ML 4.ML 12 is a hand centrifuge with buckets for fourtubes. It is described in Section 1.5.ML 13 is the Lovibond comparator, which is describedin Section 5.10. The tubes for it are ML 48d andthe discs for it ML 2 la, b, c, d, e.ML 14a, b, c, d. These are containers. A container is abottle, tube, or box for putting something into. The containershere are mostly for putting specimens into. Thefirst two are glass and have metal caps with rubber discsor hers inside. The smaller glass container is a bijoubottle, and the larger glass container is a uulversaIcontainer. ML 14c and ML 14d are containers made ofplastic and have plastic caps. The polypot is for sputumand stools and the polytube for blood. Read about themin Section 4.6.ML 15 is a double-celled counting clamber. It isused for counting cells in the blood and CSF. Lookat Sections 7.29 and 9.9. It is very carefully madeand is therefore expensive. A counting chamber costs$7.2.ML 16 is a spare cover glass for the double countingchamber ML 15.ML 17 are coverslips for ordinary microscope slides.Cover glasses for counting chambers are bought one byone and are expensive, costing $0.47 each. Coverslips formicroscope slides are bought in boxes of half an ounce.There are many coverslips in a box; so each coverslip ischeap. Cover glasses are thick; coverslips are thin. Don’tmix them up, and always keep cover glasses verycar-efuliy.ML 18 is a glass measuring cylinder. It is a highnarrow glass bottle with graduation marks on the sidelookat Picture L, FIGURE 5-2. It holds 100 ml and has aplastic stopper. You are given two cylinders in case onebreaks.ML 19 is a plastic measuring cylinder which holds1,000 ml or one litre. It therefore holds ten times asmuch as the glass measuring cylinder ML 18.ML 20 is a diamond pencil. It is like an ordinarypencil, except that it has a very small diamond instead ofa lead. A diamond is a very hard, very expensive jewel orprecious stone. A diamond can be used for writing onglass. Use it for writing on microscope slides that are tobe stained by the Ziehl-Neelsen method. You can writewith a grease pencil, but grease pencil marks come offeasily, and it is better to use a diamond.ML 2 1 is a Lovibond disc. Only one disc is shown inthis picture-the one for haemoglobin called 5/37X.This is the only one you will need in a health centre, butin a hospital you will want four more. Two are for theblood urea (5/9A and 5/9B), and two are for the bloodsugar (5/2A and 5/2B). You will not need them if youhave the Grey wedge photometer or the EEL colorimeter.ML 22 is a filter paper and the box in which you buy100 papers. There are very many kinds of paper andmany sizes. The kind you have is the ordinary kind forgeneral use and is called Whatman No. 1. You are giventwo sizes. One size is 5.5 cm in diameter, and the otheris 11 cm in diameter. The smaller one is for the bloodsugar method (Section 7.42), for the filtration method forhaemoglobins A and S, and for Fouchet’s test (Section8.8). The larger one is for filtering stains.ML 23 is a filter pump. It is a machine for sucking inair and is used for cleaning pipettes. It is described inSection 3.3. A filter pump has to be fixed to a tap. If youare to use a pump of this kind you must have runningwater and the right kind of tap. Many laboratories willnot be able to use a filter pump, because they will nothave running water under enough pressure.ML 24 is a pair of forceps. Forceps are used forholding slides or swabs which you do not want to hold inyour fingers.ML 25 is a plastic funnel for holding filter papers. It is6.3 cm across the top and holds both the 5.5 cm and the11 cm filter papers.ML 26 is a square of wire gauze (cloth) made of ironwire. In the middle of the gauze is a circle of asbestos.Asbestos is a special kind of wool which cannot be burnt.

- D0dt - mix UD these two II!6 ~~;',:yzh.,, 2 stok2Jthis is amcoverglassit is thickand expensiveqwit1 h 2sperunstoppered “‘YJ : -400. ‘psot3 _7oes_b”&

Special equipment in the main list 1 2.2The wire gauze and the asbestos do not get burnt in aflame. The wire gauze is put on top of a tripod (ML 42)when a glass beaker (cup) is being heated with a Bunsenburner. The gauze stops the glass breaking. You aretold to use a tin in Section 7.42 instead of a beaker,and a gauze square is not needed with a tin. But asquare of wire gauze goes with a tripod, and you havebeen given one in case you have a beaker and want touse it.ML 28 is a loop holder. It is a rod of aluminium with achuck at one end for holding a wire loop (see Section3.10) or a needle (see Section 11.11).ML 29 is lens tissue. This is a special kind of very softpaper for cleaning the lenses of microscopes-look atSections 6.16 and 6.17, also Picture Y, FIGURE 6-20.Lens tissue is sold in a box of 100 tissues which youmust cut up and make into booklets (little books). Youcan buy lens tissue in booklets, but it is much cheaperto buy it as a box of sheets and make the booklets yourself.ML 30 is the Olympus Model K microscope which isdescribed in Chapter Six.ML 3 1 is a grease pencil. This is a special pencil witha greasy lead which will write on glass. A ‘Chinagraph’pencil is one kind of grease pencil. You can also write onglass with a diamond pencil (ML 20) or a spirit pen (ML66), or with paint and a small paint brush (ML 56).Grease pencil writing is easily rubbed off. Spirit penwriting lasts longer but can be removed by spirit, xylol,or washing.ML 32 is a blood pipette. This is a special glass tubefor measuring small volumes of fluid. This blood pipettemeasures 0.02 ml. O-05 ml, and 0.1 ml of blood. Youwill have to use it with a rubber tube (ML 34) and a glassmouthpiece (ML 33).ML 33 is a glass mouthpiece. It is a little glass tubewhich goes into your mouth and joins on to a rubbertube. The other end of the rubber tube is fixed on to apipette. It is easier to suck fluid into a small pipette if youuse a mouthpiece and rubber tube. Look at Picture 6,FIGURE 7- 1.ML 34 is a rubber tube for the mouthpiece describedin the paragraph above.ML 35a, b, c, are graduated pipettes. These pipettesare like the blood pipette (ML 32), but they are larger.They are all described in Section 5.7. The three kinds ofpipettes listed here hold 2, 5, and 10 ml.ML 36 is a set of proteinometer standards. Proteinometermeans ‘protein measurer’. This is a row of smalltubes in a black box. Each tube is for a different amountof protein. They are used for measuring the CSF proteinand are described in Section 9.13. This set of standardswill only be needed by hospitals. If you have a Greywedge photometer, you can use it to measure the CSFprotein, so you will not need these proteinometer standards.ML 37 are microscope slides. You will use manymicroscope slides. Slides are used for making films of aspecimen.ML 38 is a spatula or spoon for taking chemicals outof bottles. A knife blade or a teaspoon can be used, but itis better to use a spatula. This spatula is made of nickelwhich is a metal that does not rust and is not easilyharmed by chemicals.ML 39 is an ordinary lamp which burns spirit-aspirit lamp. It is cheap and has been put in the list in caseyou run out of gas for your Bunsen burner. Use methylatedspirit which gives a clear hot blue flame. Don’t useparaffin which will give you a smoky cool yellow flame.Don’t use petrol because this may be dangerocs. Thislamp has a cap. Put the cap on when you are not usingthe lamp. This will stop the spirit evaporating (drying up)and will make it easier to light the lamp the next time youwant to use it. A spirit lamp is not large enough to makePasteur pipettes, and you will have to use a paraffinpressure stove (ML 71) (see Section 3.9) or a Bunsenburner.ML 40 and 4 1. You are given two kinds of test tuberack. The aluminium one holds small tubes and goes inthe water bath (ML 53). It is used in blood transfusionand will only be needed in hospitals. The other test tuberack is made of polythene and is kept on the bench. Thealuminium one holds Kahn tubas and cross-matchingtubes. The polythene one holds ordinary test tubes aswell. It has pegs (short rods) on which wet tubes can beleft to drain after washing. A test tube has been showndraining in the picture. By draining we mean lettingdrops of water run out so that air can dry the inside ofthe test tube more easily. Always wash out your testtubes when you have finished and let them dry on thepegs.The plastic test tube stand can be replaced by thewooden test tube block shown in Picture E, FIGURE3-8.ML 42 is a tripod. This is a stand with three feet(tri = three, pod = foot). It is used for holding thingswhile you heat them with a Bunsen burner. You will useit for measuring the blood sugar in Section 7.42. Thetripod holds a tin of boiling water.ML 43 is a rubber teat. Teats are little rubber bagswhich fit the glass tube ML 5 1. With a specially pointedglass tube they make a Pasteur pipette. You are told howto make Pasteur pipettes in Section 3.9.ML 44 is a thermometer (therm = heat, meter = measure).A thermometer is used to measure how hotsomething is-its hotness or temperature. The thermometerhere is a glass tube with a bulb (small empty ball)at one end. This bulb is filled with a liquid metal calledmercury which runs up and down the tube. When themercury is cold it contracts (gets smaller) and only fillsthe bulb and the bottom part of the tube. When themercury gets hot it expands (gets bigger) and moves upthe tube. The coldest this thermometer will measure isthe temperature at which water freezes or 0°C. ’ standsfor degrees and C for Centigrade. Water boils at IOO’C,and the difference between freezing and boiling is dividedinto a hundred parts. Each part is one degree. ‘Cent’means one hundred, and so we have ‘Centigrade’. This

ML 38 Spatula ML 39 Spirit lamptube closedML 41 Test tube stand,plastic to use onthe benchML 42 Tripodscrew capmercury up asfar as here20-front cut away plasticglasstube rv holes around thebulb of the thermometerML 43 Rubber teat ML 44 Thermometer ML 45 Thermometorsheath\-IML 46 Plastic centrifugetube, ungraduatedML 47 Graduatedcentrifuge tubelo(try hard to get ALL:the equipment you need !!--I ml.i63II,-- IgraduationML 48a Standard ML 48b ‘Cross ML 48c Kahn ML 48d Lovibond ML 49 Plastic tiletest tube matching tube’ tube tube with depressionsJm?rk/depressions(holes) 2Only a short lengthof the tube is shownhereML 50 Rubber tube1t 1LidML 51 Glass tubing,this is a big bundleof tubingML 52 Polythenewatch glassML 53 Electricwater bathML 54 Sparewicks for thespirit lampML 55 Nickel-chromewire for the loopholderFig. 2-3 Special equipment in the main list-three

.I‘:; ‘1 :....’ ;.,. . .I, _(Ordinary equipment in the main list 1 2.3thermometer will measure as high as 5O”C, and 50°C isat the top of the tube. It will measure 37”C, which is animportant temperature, because this is the temperature ofthe human body. A waterbath at 37°C is used for bloodtransfusion methods. You will use the thermometer tomeasure the temperature of this waterbath. Centigrade isan old word and you will often 6nd the newer wordCelsius used instead.ML 45. A glass thermometer iseasily broken. ML 45is therefore a thermometer sheath to stop your thermometergetting broken. A sh&h is a covering which protectsthe thing inside it. This sheath is a metal tube withpart of its front cutaway. There is a cap at the top whichunscrews. Unscrew the cap and gently push the thetiometerdown inside the sheath with its bulb first. Put thecap back. Turn the thermometer round so that you cansee the graduation marks on the scale. Always keep thethermometer in its sheath. It will be safer this way. But,even so, be careful not to drop it. A thermometer can stillbreak, even inside its sheath.ML 46 and 47 are plain and graduated centrifugetubes. They are made of polypropylene or nylon, whichare strong kinds of plastic. They have conical bottomsand are made to go into the buckets of a centrifuge. Onetube is plain and the other has graduation marks on it upto 10 ml. These centrifuge tubes are described inSections 1.5 and 5.7.ML 48a-d are test tubes. A test tube is a short glasstube with thin sides and a round bottom. You are givenfour kinds of test tubes. ML 48a is tbe ordinary test tubeand is most often used for testing the urine. ML 48b is ashort narrow test tube which is only used for cross matchingblood-look at Section 12.6. In this book it is calleda cross-matching tube. ML 48c is a very useful size oftube in which to ‘wash’ red cells for blood grouping, andit is called a Kahn tube. ML 48d is the special Lovibondtube or cell. It is square and fits the newer ‘Lovibond1000’ comparator. The older kind of Lovibond comparatorhad round tubes (ML 48e). When getting tubes foryour Lovibond comparator, make sure you get the rightones. These tubes are expensive and the square kind costabout $1.1 each. Both tubes have the word LOVIBONDwritten on them and must not be used for anythingexcept comparing liquids in the comparator. Never heata Lovibond tube, or put it in a centrifuge.ML 49 is a tile or spotting plate. It is a square of whiteplastic with some shallow holes in it. It is used for bloodgrouping and for testing the urine for INH (Section 8.9).A white builders’ tile can also be used for blood grouping,so can a piece of plastic sheet, such as the clear kindof plastic ca!led ‘Perspex’.ML 50 is two metres of rubber tube. This is for thewater pump and the Bunsen burner. You may be luckyand have running water and the right tap to make itwork-see Picture F, FIGURE 3- 1.ML 5 1 is a parcel of many long pieces of glass tube.This is the glass tube from which you can make thePasteur pipettes described in Section 3.9. It is between 6and 7 mm across. This is the external or outside dia-meter, and each piece is about one and a half metres long.Glass tubing is bought by the kilo. You are given twokilos of glass tubing.ML 52 is a polythene watch glass. There is a roundglass on the front of a watch. This is a watch glass. Thewatch glasses you are given are much bigger than thoseon wrist-watches and are made of plastic, not glass. Theyare really dishes or plates. They are used for weighingchemicals on a balance.ML 53 is an electric water bath. It is made so that itwill keep water at any temperature between that of aroom (about 20°C) and 100°C (boiling temperature orboiling point). You will use it at 37°C.ML 54 are spare wicks for the spirit lamp ML 39. Awick is the cloth in a lamp that is soaked in oil or spiritand bums at one end.ML 55 is wire for making loops which go in the loopholderML 28. Section 3.10 tells you how to make theseloops. It is called nickel-chrome wire because it is madeof two metals which are rather like iron called nickel andchromium. Wire of this kind is not easily burnt away in aBunsen burner. This wire is called ‘22 SWG’ nickelchrome wire. 22 SWG measures how thick it is. SWGmeans Standard Wire Gauge.2.3 Ordinary equipment in the main list (FIGURE2-4)So far all the equipment that we have described has beenequipment that has to be ordered specially. But there aremany ordinary things, like cups and jars which are usefulin a laboratory. These can be bought in most ordinaryshops. You may say that it is not worth drawing picturesof all these things. Perhaps it is not, but it may help youto get them. This ordinary equipment is just as importantas the special laboratory equipment.ML 56 is a small paint brush. It is used with the quickdrying paint to label bottles as is shown in Picture C,FIGURE 3-8. A spirit pen can be used, but paint looksmuch better. Carefully labelled bottles will help you tobe proud of your laboratory.ML 57 is a bucket with a handle. You will want six ofthese. Two of them should be metal. They are shownunder the bench in FIGURE 3- 11.ML 58 are ordinary plastic cups with handles. Theycan be used instead of glass beakers for everythingexcept heating. Because they are plastic they do notbreak as easily as glass beakers.ML 60 is a gas cylinder. This is a big iron bottle ofgas. It is used to provide gas for a Bunsen burner asshown in Picture A, FIGURE 3-2. There are many shapesand sizes of cylinders, and you will have the kind whichis sold in your country. The cylinder must have amachine on it called a reducing valve. This lets the gasout of the cylinder slowly and is shown in Picture F,FIGURE 3-2. The reducing valve is joined to the Bunsenburner by a piece of rubber tubing (ML 50a). You willneed two cylinders. Keep one in use. As soon as it is

. . ,, ,.‘,_,I,I: ,.:, ‘Y., ,Y,,. ” ;_,/,: .’_’ 2 t Eql ripment and Chemicalsiemp@ send it away to bej filled up. While it is being filledup use ti he second one.ML 61 is ahammer. 1 ‘ou will find it very useful in alaboratory.ML 62 is a glass jar with a wide mouth and a plasticscrew cap. They are for Field’s stains, A and B, and forRothera’s reagent. These are very necessary, and if theycannot be bought locally they may have to be ordered..f&l. WLL~ cl- UIC cqu~pmcn~^^__.’ _-^_ A 1-IIIALmt:man--Ilist.1-LML 63 is an oilstone for sharpening the needleson blood transfusion equipment. It can also be used tosharpen other needles.ML 64a, b, and c are small tins of quick-dryingenamel paint which are used to label bottles and polytubeswith paint brush ML 56. You want three colours:black for bottle labels and red and yellow for polytubes-see Section 4.6.ML 65 is a metal pan with two handles and is used for22EJ140~@1ML 56 Small paintbrushML 57 PlasticbucketML 56 Plastic cupML60 Cylinder of gasand a reducing valve(not shown here)ML 61 Hammerthese wide mouthed bottlesmay have to be orderedspecially6you will want threecolours of paint,black, yellow and redwide mouth120 mlML 62mouthedwidebottleML 63 OilstoneML 64 PaintML 65 Pan with twohandlesML 66 Spiritpenall these things are just as importantthe special laboratory equipment!!asML 67 Plasticine ML 68 Pliers ML 69 PressurecookerFig. 2-4 Ordinary equipment in the main listML70 ScissorsML 71 Paraffin pressurestove and the spares togo with it

Chemicals 1 2.4heating things on the pressure stove. It is especiallyuseful for boiling slides that are covered with oil.ML 66 is a spirit pen for marking. It is useful formarking bottles.ML 67 is plasticine. This is a special kind of claywhich does not dry up. It is useful for fixing and holdingthings in a laboratory. It makes a good slide holder if youhave not got the wooden one shown in Picture F, FIGURE3-8.ML 68 is a pair of pliers. These are used for makingwire loops and are very useful in the laboratory.ML 69 is the Prestige ‘Hi-Dome’ pressure cooker. It isdescribed in Section 1.21. You should have a spare gasketand a spare safety plug for your cooker.ML 70 is a pair of scissors.ML 71 is a paraffin pressure stove; it is often calleda ‘Primus stove’. Pasteur pipettes can be made on aparffi pressure stove if there is no gas. With the stovethere should be a bottle for spirit and some ‘prickers’ forkeeping the jet clear. The jet is the little hole out of whichthe paraflin vapour (gas) comes.You will also want syringes, needles, conical urinespecimen glasses (as in Picture 3, FIGURE 8-6), cottonwool, surgical gauze, lysol, pa&in, and matches, rubberstamps (see Section 4.2), and a stamp pad. The urinespecimen glasses are ordinary hospital equipment. Glassbreaks easily, so plastic ones are better.2.4 ChemicalsYou will need about forty chemicals. These are listed inSection 13.10.The following chemicals are white powders, many ofwhich look like one another. They are: tartaric acid,salicylsulphonic acid (also called sulphosalicylic acid),barium chloride, barium peroxide, ortho-tolidine (alsocalled o-tolidine), sequestrene (this is also called sequestricacid potassium salt, and EDTA or ethylene-diaminetetra-acetic-acidpotassium salt), potassium fluoride,potassium iodide, sodium acetate, sodium carbonate,sodium chloride, sodium citrate, and the two kinds ofpotassium phosphate. Trichloracetic acid easily takeswater out of the air. It is said to be hygroscopic, and itmay even take enough to make a solution. Keep the lidon carefully to stop this. Copper sulphate forms beautifulblue crystals or big blue lumps. Ferric chloride formsdark brown crystals. It too is hygroscopic. Iodine formssmall deep brown-black crystals. Phenol forms crystalswhen it is cold, but when the weather is warm phenolforms an oily liquid. Sodium chloride is the ordinarytable salt that we ?at with our food. Sodium hydroxide isbest bought as small pellets (little balls). It is very hygroscopicindeed. If you leave the lid off the bottle, sodiumhydroxide quickly forms a hard lump and is difficult toget out of the bottle. Keep the lid on.The several kinds of phosphate are more difficult tounderstand. There are two kinds of potassium phosphatein the list of chemicals. There is di-potassium hydrogenphosphate which is written K*HPO,. K stands forKalium, which is another name for potassium, H standsfor hydrogen, and PO4 for phosphate. Notice the z afterthe K. Di means two. There is also potassium dihydrogenphosphate which is written KH2P0,. Notice the 2 afterthe H in this phosphate.In the same way there are two main kinds of sodiumphosphate. There is disodium hydrogen phosphateNa,HPO,, and there is sodium dihydrogen phosphateNaH,PO,. Na stands for Natrium which is another namefor sodium. These two sodium phosphates are not in thelist of chemicals, but you may have them, and they canbe used instead of potassium phosphates for somemethods.But difficulties do not end there. Both sodium phosphatescan be anyhdrous or without water in them(‘an’ means without, ‘hydrous’ means watery). Theseanhydrous phosphates are written Na,HPO, andNaH,PO,. They can also have different amounts of waterin them. The chemical way of writing water is H,O. The differenthydrated (watery) phosphates are Na,HP0,.2H,Owhich has some water, Na$IPO,.l2H,O which has alot of water, and NaH,P0,.2H,O which also has somewater.One kind of disodium phosphate can usually be usedinstead of another kind of disodium phosphate, but youwill want a different weight of it, depending on howmuch water there is in it. Similarly one kind of sodiumdihydrogen phosphate can usually be used instead ofanother kind of dihydrogen phosphate. You will alsoneed a different weight of it, depending on how muchwater there is in it. But a disodium phosphate will neverwork instead of a dihydrogen phosphata. Nor will adihydrogen phosphate do instead of a disodium phosphate.The right kinds of sodium and potassium phosphatescan however bc used together. Read the methodand the labei on the bottle carefully, and use only theright kind of phosphate. Section 3.20 describes howthese phosphates are used to make buffer solutions.Acetic acid, hydrochloric acid, sulphuric acid, formaldehyde(also called formalin). ‘Teepol’, methanol, spiritand xylol (also called xylene), and ether are all liquids. Ifyou have no ether, petrol can be used instead for themethod in Section 10.3. Most of these liquids havesmells which you will soon come to know.Concentrated hydrochloric acid and concentrated sulphuricacid are dangerous. Don’t pipette them with apipette in your mouth. If you get them on your hands,wash your hands immediately. Never add water to sulphuricacid. The mixture will get hot and spit. It willblind you if it gets in your eyes. When you dilute sulphuricacid, add sulphuric acid gently to water. There aremore instructions for doing this in Section 3.26.Methanol is the same as methyl alcohol. Ethanol is thesame as ethyl alcohol. Ethyl alcohol is the alcohol inbeer; methyl alcohol is very poisonous. ‘Spirit’ is about95% ethyl alcohol and 5% water. There are several kindsof spirit, some have poisonous chemicals (includingmethyl alcohol) added to them to stop people drinking

2 1 Equipment and Chemicalsthem. The purple methylated spirit is spirit of this kind.Surgical spirit is a purer kind of spirit, but it too ispoisonous. When you read about ‘spirit’ in this book, thismeans either methylated spirit or surgical spirit. It isbetter to use surgical spirit. Spirit is a mild antiseptic anda useful fuel. It is used in the spirit lamp. Absolutealcohol is pure ethyl alcohol with no water and nopoisonous chemical in it. It is very expensive, and it isnot used for the methods in this book. But, if you haveabsolute alcohol it can be used for any of the methodsthat require spirit.Alcohol, acetone, spirit of any kind, and xylene are allinflammable: they will bum like petrol. TAKE CARE.Ether is especially inflammable, so take very special care.Never put ether near a flame. Never have ether on thesame bench as a flame, because the vapour (gas) fromether may roll along the bench and cause a fire or anexplosion (a big bang). Besides being inflammable theseliquids are also very volatile. This means that they easilyevaporate, disappear and go into the air if the top is leftoff the bottle. Ether is especially volatile. KEEP THECAP ON.Ammonia is a very dangerous liquid with a verystrong burning smell. A little ammonia in air or watercan spoil Nessler’s solution and the blood urea method,so keep ammonia and Nessler’s solution well apart (seeSection 7.4 1).You will want the following dry stains. They are alldeeply coloured crystals-basic fuchsin (purple, a mixtureof blue and red), brilliant cresyl blue, crystal violet (violetis a kind of very deep blue), Field’s stain A (blue), Field’sstain B (red), malachite green, and methylene blue.Immersion oil is a special thick, clear oil. The 250 mlyou are given is to fill up the oil bottle that is suppliedwith your Olympus microscope. You can also make anoil bottle (see Section 6.7).You should have some universal indicator test paperand also a bottle of ‘Acetest’ test tablets for testing acetonein the urine (see Section 8.7).The last group of chemicals are called biologicals.These are chemicals which come from living organisms,such as man or animals. All biologicals must be kept in arefrigerator. If they are not stored in the cold, they soonstop working. Bacteria may grow in them, and they mayputrefy (see Section 1.15). The biologicals you will needare anti-A serum, anti-B serum, and bovine albumen.These may be given you as powders or as liquids. Theywill not be wanted in a health centre. KEEP THEM INA REFRIGERATOR.2.5 The choicesSo far we have described only the main list of equipment.Some people may want to change this list. The changesthey might want have been described here as choices.The methods for all these choices are described in laterchapters. You can find out more about this equipment inthe sections from 13.12 onwards.What can you do if you cannot get everything youwant? Most things are essential-you have got to havethem. But there are some things for which there aresubstitutes (a substitute is something which will doinstead of something else). Petrol (it is sometimes calledgasoline) can be substituted for ether in the formol+ztherconcentration test. Liquid paraffin, which is often used inhospitals, works nearly as well as immersion oil. Softtoilet paper can be used instead of lens paper. Smallsquares of ‘Cellophane’ or polythene can be used insteadof coverslips. None of these things, however, work aswell as the right thing!Choice 1. Replacing the Lovibond comparator bythe Grey wedge photometerIf you have the Grey wedge photometer you will notneed the Lovibond comparator or its tubes and discs.You will not need the set of proteinometer standards(ML 36) either. Read about the Grey wedge photometerin Section 5.11.Make sure you have spare bulbs and spare cells-seeSection 13.13.Choice 2. Replacing the Lovibond comparator withthe EEL calorimeterYou will not need any of the Lovibond equipment, butyou will need the proteinometer standards set. You willwant all the EEL equipment you see in FIGURE 5-13.You will want the three Ilford filters 625, 608, and 622,a set of EEL tubes, and preferably a spare selenium cell.You will also need one of the standards for haemoglobindescribed in Section 5.19. There are other spares alsowhich may be needed-see Section ! 3.15.Choice 3. Silica gelIn very warm wet countries fungi grow on the lenses ofmicroscopes and spoil them. Microscopes should thus bekept in a tightly closed box or bag with silica gel to takewater out of the air and keep them dry, so that fungicannot grow on them. See Section 6.18.Choice 4. Electric centrifugesThere is a hand centrifuge (ML 12) in the main list ofequipment. If you have electricity there are several electriccentrifuges you can use. There is a cheap plastic onecalled the ‘MSE Minette’. There is also a bigger onecalled the ‘MSE Minor’ on which.you can do the microhaematocrit.There is also a special one, the ‘Model X’,which will run on 220-volt main electricity and also on acar battery. You may also have the Bickerton-Eavesmicrohaematocrit centrifuge.Choice 5. A deep counting chamberCounting chambers are discussed in Section 7.29. In themain list there is a Neubauer double-celled counting

Stock and spares 1 2.6chamber 0.1 mm deep. But it is also useful to have aFuchs-Rosenthal chamber O-2 mm deep.Choice 6. Testing the urine for INHIt is sometimes useful to be able to test the urine oftuberculous patients for the drug called INH. Twoextra chemicals are needed, chloramine-T and potassiumcyanide. Potassium cyanide is so poisonous (so likely tokill you if you eat it) that it has not been put on the mainlist. Read about the care you must take in using it inSection 8.9.ChoQe 7. The cyanmethaemoglobin methodThis is a more accurate way of measuring haemoglobin,the equipment for it being listed in Section 13.20.Choice 8. The use of sodium azideSodium azide can be used to preserve (keep) samples ofserum to be sent to a central laboratory (see Section 4.10).Choice 9. Using ammonia to measure the haemoglobinIt is usual to use ammonia rather than sodium carbonatefor measuring the haemoglobin.Choice 11. Dichromate cleaning fluidMost glassware can be cleaned with soap and water. Butif it is very dirty a mixture of potassium dichromate andsulphuric acid will help to clean it. Sulphuric acid isdangerous and caustic. Sections 3.12 and 3.26 will tellyou how to make and use dichromate cleaning fluid.Choice 12. Making some reagents locallySome reagents are best made up in a central laboratoryand given to you ready made. Section 13.25 will tell youthe chemicals you need as well as how to make thesereagents if you have to.Choice13. ‘Dextrostix’This is a special paper test for measuring blood sugar. Itis very quick and easy to use, but it is not accurateenough to replace the method in Section 7.42.Choice 14. RotheraS testThe older and slightly cheaper way to test for acetone inthe urine is to use a mixture of sodium nitroprusside andammonium sulphate. This is Rothera’s method. The‘Acetest’ tablet method is in the main list, but you maywant to do Rothera’s method. Both methods are describedin Section 8.7. Sodium nitroprusside forms deeppurple crystals.Choice 15. ‘Ictotest’ tabletsThese tablets are used to test the urine for bile pigment.Fouchet’s method is the older way of doing this: and thechemicals needed for it are in the main list (see Section8.8).Choice 16. The *Cellophane’ thick stool smearThe materials for this very useful way of examining thestools for helm&h ova are described in Section 13.29.Choice17. ‘Labogaz’Gas is being used more and more often and can bebought in tins that can be used with a special kind ofBunsen burner. ‘Labogaz’ is gas of this kind and isdescribed in Section 3.4.Choice18. This bookThe easiest way to make sure that the people who need toread this book can do so may be to put it in the equipmentlist and give it an ML number. It is thereforeincluded here as Choice 18 and called ML 15 1.The choices you will use in your hospital or healthcentre will be decided by the people in charge of yourmedical service. There is a table in Section 13.32 wherethese choices can be written down.2.6 Stock and sparesTry very hard to keep enough chemicals to last you atleast 6 months. Don’t wait until something is finished 1before ordering some more. You should always haveenough of anything in stock so that if any order fails tocome you have time to order again before running out.Note how much of a thing you use, and take special careover things that are soon finished, such as spirit, gas,paraflln, immersion oil, and slides.Some equipment needs spare parts. For example, keepa spare gasket for the pressure cooker, spare bulbs forthe EEL, and a spare cell for the Grey wedge. Never relyon one of anything that is easily broken, one pipette forexample. Make a list of all the spares you need, and tryto get them-NOW.QUESTlONS1. What kinds of bottle do you know? Describe oneuse for each kind.2. What kinds of pipette do you know? How are theyused?3. What is the difference between a cover glass and acoverslip?

7,i’ .I2 1 Equipment and ChemicalsI‘_,4. A spirit lamp is easy to use, but how would YOUlook after it? .5. What figures are there on your thermometer, andwhat do they mean?6. What different kinds of phosphate are there, andhow do they differ?7. What do copper sulphate, iodine, ferric chloride,sodium nitroprusside, and phenol look like?8. Formaldehyde, xylol, methanol, and ethanol allhave other names. Do you know them?9. What are the differences between absolute alcohol,surgical spirit, and methylated spirit?10. What specially important things must you rememberwhen you are working with (a) ether, (6) potassiumcyanide, (c) sulphuric acid?

3.1 Benches and shelvesThere are many things to do before specimens can belooked at in a laboratory. This chapter tells you how todo them.The first thing to think about is a room for the laboratory.This should be a special room where only laboratorywork is done, but, if you have no special room, youcan use part of a room which is used for something else.Many health centres have no special room for a laboratory;but almost every health centre can fmd space for ashort bench, and there is always plenty of room forshelves. An important part of a laboratory is its bench.This is a big table and is shown in FIGURE 3-l 1. Thebench must be wide enough and the right height. 75 cmis a good width and is also a good height for a bench. Ifyou have not got a bench use a table. You can do all thetests described here on quite a short bench or table, butyou must have plenty of shelves. on which to put theequipment. Keep everything yoz‘ can on shelves so thatyour bench or table is clear to work on. Just above thebench the shelves should be narrow, say, 15 cm for thefirst shelf. Further above the bench shelves can be wider,say, 20 cm for the second shelf and 30 cm for the third.The first shelf should be 30 cm above the bench and thethree shelves 45 cm apart. Under the bench there is roomfor a drawer, and there is also space for another shelf ifyou want one. The length of the bench needed dependsupon how many laboratory workers there are. Eachworker only needs about 6 feet of bench.Before you use a new bench always see that it is wellcovered with polish. If you have nothing else use a mixtureof candle wax and parat&. A well-polished benchwill not stain or get marked so easily.bench with two pipettes across it joined by rubber tubes.A four-gallon petrol tin cut in half can also be used, asshown in Picture B. If a tin or bucket is used for stainingslides it will only fill up slowly.Put two pieces of glass tube (ML 5 1) across the sink,the tin or the bucket to make a staining rack for slides.These tubes are to hold slides while they are beingstained and must be about two inches apart. If you wantto know how to cut glass tube, look at Section 3.9. Thesetubes must be horizontal, so that the stain is the samedepth all over the slide, as shown in Picture A, FIGURE3- 1. The best way to hold these tubes is to use specialmetal bars with levelling screws which rest on the benchat each side of the sink. These are shown in Picture F,but they are not really necessary. You can use stickingplaster (surgical adhesive tape or ‘strapping’) or awooden block, or two nails. If you are using a tin as asink, cut the edges of the tin so that the tubes lie flat asshown in Picture B. You will have to borrow a pair of tinshears (tin scissors) to cut the tin. With these cut holes ornotches in the edge of the tin so thatthe tubes can rest inthem and lie flat. Cut away the front of the tin so that youcan get a spirit lamp or a lighted swab underneath theslides to warm them in the Ziehl-Neelsen method (seeSection 11.1). If you use a plastic bucket, cut the edge ofthe bucket with a knife in the same way so that there is aplace for the tubes to lie in. Stick the rods to the bucketwith a piece of sticking plaster as in Picture D. If youhave many slides to stain by the Ziehl-Neelsen methodyou may find it useful to put more than two bars acrossyour sink or your tin.You can wash up dirty glassware (anything made ofglass) in any sink. Washing up can also be done in abucket out-of-doors.3.2 Sinks and staining (FIGURE 3- 1)You will want two sinks, one on the bench for stainingand one somewhere else for washing up specimen bottles,etc. The best kind of sink for staining is the specialsquare laboratory one that you will see in Picture F. Butyou can use a bucket as shown in Picture C and inFI'JURE 3- 11. Some people use an enamel basin set in the3.3 Water (FIGURE 3- 1)You must have water, but it need not be running water.All the methods in this book can be done with waterfrom a bucket. Underneath the bench in FIGURE 3.11 aretwo buckets, one is for clean water and the other is fordirty water. Water is taken from the clean bucket with a

3 i Making the Laboratory Readyslide being stained/on a staining rack/slides covered with staina staining sink has been madefrom a plastic bucketeglass tubethe glass tubes areperfectly horizontaland the slide is evenlycovered with staina staining sink has beenmade out of an old petroltin cut in halfML 57HOW A FILTER PUMP WORKSwaterDEthis isa siphonnarrownozzleQll~,/+inlet tube4 piece has been cutz _Ifrom the edge OT mebucket for the tubeshelfwater rushes\ stone at the bottomof the waterwater comingdownII- rubber tube,use the tubefrom bloodtransfusionsets 8’bubbles ___C $1I IhiOlbl!I IAllthe glass tubesof the stainingIvrackIwater _ tglass tubesthis is the slidebeing washedSpecial levellingscrew-- - -- -J[ide covered - -with stainbenchFig. 3-lSinks and staining- -a blood pipette%bcup-LzPbeing cleanedjug and poured over the slides. A wash bottle filled withwater is also useful,Another way of getting water to a sink for staining isto use a bucket on a high shelf, a siphon, and a clip, asshown in Picture E. A siphon is a way of making waterrun upwards before making it run a longer way downwards.The flow of water in the siphon has to be started.The best way to start it is to put all the tube under thewater in the bucket and to fill it with water by moving itabout in the bucket until no more air bubbles out. Thenpinch (close) the lower end of the tube and quickly bringit over the side and well below the bottom of the bucket.Stop pinching the tube and water will start flowing. Youcan then put on the clip. Water will start flowing orice

P,,,.” y- ,.* 9.; _, ,,.Bottled gas 1 3.4more as soon as you open it again. Use the clip from ablood transfusion set if you cannot get a proper Mohr’sclip. Fix the top end of the tube in the bottom of thebucket by tying it to a stone.A better way of getting water to your staining rack isto use an aspirator jar. This is a very big bottle with a tapon the bottom to which you can fix a rubber tube. Put theaspirator jar on a shelf above the staining rack. A tinwith a nozzle soldered on can also be used.The best way of getting water to the staining sink is touse a high double tap. This is shown in Picture F. One ofthe double taps can be fitted with a piece of rubber tubewhich hangs over the slides on the staining rack. Thisrubber tube can be moved so as to wash any of the slides.A filter pump (ML 23) can be fitted to the second of thetwo taps. Push a piece of rubber tube on to the tap andon to the top of the filter pump. Push another piece ofrubber tube on to the outlet tube at the bottom of thepump. Filter pumps suck better if there is a piece ofrubber tube fixed to the outlet tube in this way. Push athird piece of rubber tube on to the side tube of thepump. When you turn on the tap and put your fingerover the end of this tube, you will feel the air sucking in.You can now put a pipette on to the end of this rubbertube and dip it into a cup of water. Water will rush up thepipette and clean it. This is a very good way of cleaningpipettes. All this is shown in Picture F.A filter pump can be used to dry blood pipettes bysucking water through them, and then sucking a littleacetone to remove the water. Acetone washes away thewater and then rapidly evaporates as air is suckedthrough. This leaves the pipette dry. Dry pipettes measureblood more accurately than wet ones. If your rubbertube is too big to fit a small blood pipette, tie a knot inthe end of the tube and then push the end of the bloodpipette through a small hole in the tube just above theknot.Picture G shows you how a filter pump works. Waterunder pressure comes out of a narrow nozzle and iscaught in a small funnel. As it rushes from the nozzle tothe funnel it catches and traps air. This sucks air fromaround the nozzle and so from the side tube.The side tube has a valve on it which is not shown inPicture G. This valve is made from a small rubber ballon the end of a pin. This ball may swell and stick andstop the pump working. When this happens unscrew thenut on the side tube and take out the rubber ball with itspin. For most uses the pump works perfectly wellwithout its valve. Put the valve back with the pin thesame way round as it came out.3.4 Bottled gas (FIGURE 3-2)The Bunsen burner (ML 10) burns bottled gas. There areseveral kinds of bottled gas each with different cylindersand valves (taps). We shall describe ‘Afrigas’. OTHERKINDS OF CYLINDERS AND VALVES AREDIFFERENT FROM THOSE FOR AFRIGAS, SO BECAREFUL. Afrigas can be bought in small metal bottlesor in big cylinders. One of these big cylinders and aBunsen burner are shown in Picture A. The gas in thecylinder is under pressure (it is very tightly pushed intothe cylinder). So a special valve, called a reducing valve,has to be used to let the gas out of the cylinder slowly. InPicture I you will see a reducing valve tixed to the top ofa cylinder. Pictures E and F show the top of the cylinderand the reducing valve separately. The reducing valve isfixed to the top of the cylinder with a big nut (a nut issomething that turns on a screw). When the reducingvalve is not fixed to the cylinder, a cap is fixed to itinstead-look at Picture D. This cap keeps dirt out ofthe place where the reducing valve fits. There is a tap onthe top of the cylinder to turn the gas on and off. A littlerod sticks out and fits a key. Turned to theleft the tap ison; turned to the right the tap is ofl.In Picture A the Bunsen burner has been shown fixedstraight to the cylinder with a rubber tube. Very often acylinder is tixed to metal pipes which go under a benchand bring the gas to taps on the top of the bench. Gas canthen be turned on and off at these taps on the bench.Wherever possible cylinders of gas should be outside abuilding so that if gas escapes it will blow away and notcause a fire. When gas escapes inside the laboratory itwill mix with air. If there is a light in the laboratory thismixture of gas and air may explode and destroy thelaboratory.Gas may leak slowly from pipes and valves, so it iswise to turn off gas at the cylinder at the end of a day’swork. Gas smells and you can usually smell it in a roomif there is a leak. Look out for the smell of gas, and ifnecessary find the leak.METHODCHANGING A CYLINDER OF ‘AFRIGAS’ FIGURE 3-2MAKE SURE THAT THERE ARE NO FLAMESAROUND WHEN YOU CHANGE A CYLINDER.Use the special key to turn the tap of the cylinder tothe right-OFF.Unscrew the nut on the bottom of the reducing valve,and take the valve off the cylinder.Make quite sure that the tap of the new cylinder isalso turned to the right-OFF.Unscrew the cap on the new cylinder.Make sure that the tap on the cylinder and the placewhere the reducing valve fits it are both clean. if necessary.fit a new washer.Screw the nut on the reducing valve into the tap onthe new cylinder.Turn the tap on the cylinder to the left-ON. Gas willcome out of the reducing valve.‘Labogaz’This is another useful, but more expensive kind of gas. Itis shown at the bottom of FIGURE 3-2. The gas comes in

moky001ilentlameYred hota largecylinder kof bottledQas Y-ML50turned so thatthe hole isopen and gasmixed withair is burning0,ML60-reducing valvealveL hang up the spanner anail through this holetop ot cylmderTHESE THREE PICTURES SHOW THE BURNER‘Labogaz’WHEN IT HAS BEEN TAKEN TO PIECES‘---Bunsen burner3 place where the Bunsen HOLDER-h&s .._.I fnr .-. air -.. h,,mnr C,..n..le i” . -collar fixing - to mix with gasscrewgas adjustingscrewBUNSENBURNERmholderthe spike is in hereit comes out to make ahole in the tin as theBunsen burner is screwedinFig. 3-2Gas and the Bunsen burner

The Bunsen burner 1 3.5the tin drawn in Picture L. These tins are full of liquidgas under high pressure. They fit into the holder drawnin Picture M and are held by the arms. The Bunsenburner drawn in Picture N screws into the top of theholder. At the bottom of the Bunsen burner is a blackrubber washer, and in the middle of this washer is asharp steel spike or point. As the Bunsen burner isscrewed into the holder the spike comes out of thewasher and makes a hole in the tin. The rubber washerstops the gas getting out and keeps it inside the burner.The gas adjusting screw adjusts the height of the &meand turns the burner off completely when it is not beingused. The collar has a hole in it to let air mix with thegas, just as in the Bunsen burner described in the nextsection. The collar is fixed with a collar-fixing screw.There are three things to remember in using the‘Labogaz’ burner.ALWAYS UNSCREW THE BURNER BEFOREFI-ITING A NEW TIN OF GAS INTO THEHOLDER. Screw the Bunsen burner into the holderafter the tin has been put inside it. If the Bunsen burner isscrewed into the holder before you put the tin of gas intoit, all the gas will come out. It may catch alight and bevery dangerous.DON’T TAKE A TIN OUT OF THE HOLDERUNTIL THE TIN IS EMPTY. DON’T UNSCREWTHE BUNSEN BURNER UNTIL THE TIN ISEMPTY.3.5 The Bunsen burner (FIGURE 3-2)Look for the Bunsen burner in Pictures A, B, and C. Youwill see that it has a metal collar with a hole in it. Acollar is something that goes round something, just as thecollar of a shirt goes round your neck. This collar can beturned round the main tube of the Bunsen burner. At oneposition (place) the hole in the collar comes opposite ahole in the main tube of the burner. This hole lets air gointo the main tube of the burner to mix with gas comingfrom the cylinder. The mixture of air and gas bums atthe top of the main tube. Picture B shows the Bunsenburning with the hole shut. No air is getting to the maintube, and the pure gas is burning with a tall, smoky,yellow, silent, cool flame. Picture C shows the collarturned with the hole open. Air is getting into the maintube, and the mixture of air and gas is burning with ashort, smokeless, clear, noisy, hot flame. If the hole ishalf open the burner will bc silent, but otherwise theflame will be like that in Picture C. Adjust your Bunsenburner by turning the collar until the flame is hot, clean(no smoke), and sileht.In Picture C you will see that there are two wire loopsin the flame. These loops are described in Section 3.10.Loop Y is near the bottom of the flame, and you will seethat there is a cool piece of the wire in the middle of theflame. This part of the loop is cool because it is in thecool mixture of gas and air which has not yet burned. Atboth sides of this cool piece a short piece of the wire isred hot. These red hot places have\ been drawn deeplyblack in this picture.Loop X is sloping and is higher up in the flame of theBunsen burner. It is red hot right through, and there isno cool place in the middle. Whenever you flame yourloop. put it towards the top of the Bunsen flame whereloop X is, or a bit higher. You will read more about howto flame your loop in Section 3.10..3.6 ElectricityIt is useful to have mains electricity to provide light forthe laboratory. Mains electricity is electricity whichcomes from outside the laboratory. As well as lightingthe laboratory, it can be used to provide light for amicroscope lamp, a Grey wedge photometer, an EELcalorimeter, or an electric centrifuge. Many laboratorieswill not have mains electricity and will have to get electricityfrom a car battery instead. This can also be u&dto run an electric centrifuge (Model X, ML 12 1, Choice4, Section 13.17). Microscope lamps are described inSection 6.11 and the Grey wedge photometer in Section5.11.3.7 Using the battery of a Land-Rover (FIGURE 3-3)The easiest way to use the electricity of a car battery is totake it from the battery of a car which is standing still.This is especially useful when a Land-Rover takes adoctor or medical assistant to hold a clinic at a distantplace-a mobile (moving) clinic. The Land-Rover hasspecial sockets (holes) on its dashboard where metalplugs can be put in to use the electriclcy of the 12 voltbattery. A plug-is something which can be pushed into ahole. The dashboard is the place where the instrumentsare in a car. A volt is a measure of the strength ofelectricity. A microscope or a centrifuge can thus beused very easily at a mobile clinic. Special plugs can bebought, but the bare ends of wire can be pushed into thesesockets and held there with matchsticks. FIGURE 3-3shows the sockets on the dashboard of a Land-Rover.Plugs have been pushed into the sockets, and wires areleading outside the window into a hut. The driver of theLand-Rover is using an Olympus microscope to look atthe stools for hookworm. He is using a 12-volt bulb inhis microscope lamp (ML 30k). The medical assistantand the two nurses he brought with him are looking atthe patients.The battery of any car can be used, but the wires willhave to be joined up specially because most cars do nothave sockets on their dashboards.3.6 Using spare car batteries. The Honda generatorIt may be impossible to use the battery of a car which isstanding still. But it may be possible to use a spare

3 1 Making the Laboratory Readythe plugs have been pushed intosockets on the dashboard\ Isteering wheelthese wires are going outside the window of the Landrover ‘dashboardand are taking electricity to the lamp of a tiicroscopeFig. 3-3 Using the battery of a Land-Roverbattery and charge (fill) it in a car while it is moving.Eattteries can also be charged with a trickle charger, or aHc r aa generator.A car can be used to charge a battery by first making aspecial place for a second battery. The car can be startedon its own battery and then changed over to the secondbattery while it is running. This second battery can thenbe charged during a journey. A garage may be able to fita place for a second battery together with the wires andswitch for charging it.A trickle charger is a machine which will charge abattery using mains electricity. Many hospitals havemains electricity for part of the day only. A battery c;anbe charged with a trickle charger during the time that themains electricity is on. When the mains are off the electricitythat has been stored in the battery can be used.Remember to turn off a trickle charger as soon as themains electricity is shut off. If this is not done the batterymay start discharging (emptying) itself.A Honda generator is a small petrol engine that generates(makes) electricity. The smallest generator makes alittle ‘mains’ electricity (220 volts AC) for lighting a fewlamps. It can also make 12 volt (DC) electricity forcharging a battery. Honda generators are not expensiveand may be the easiest way of providing a laboratorywith electricity. However, if they are going to last a longtime Honda generators must be looked after very carefully.Read the instruction book with great care.Car batteries also need looking after. They are filledwith dilute sulphuric acid, which slowly dries up. Theymust be ‘topped up’ (jilled) to just above the plates withdistilled water (see Section 3.15) once a week. If ordin-ary water is used batteries will slowly spoil. If you haveno distilled water use rainwater.Batteries must always be recharged as soon as theyare empty. If they are left discharged for any time theywill hold less electricity next time they are charged. Inthis way they are soon spoiled.3.9 Making and using Pasteur pipettesA pipette is a glass pipe or tube which is used to take alittle liquid from one place to another. One of the mostuseful pipettes is named after a great Frenchman, LouisPasteur-the ‘Pasteur pipette’. Pasteur pipettes are madeFig. 3-4The Honda generator12 volts DC or220 volts AC

.,;-./I ,__( .if you have no diamondpencil you can usean amwule file todiamondpencil 1 I I I I l/kdiamond \ /Lglass tube L-scratch-)II I I hbld the tube iike \I I Ihe Qunsen bylrner --this tube has beenin the flame solong that its endhas become round1 \I1 ”” 14turn the tube roundand round while itis being heated\ass get really soft!!you pull. Pull steadilycut here with your,I diamond pencil \this pipette is readyto be sterilizedcotton woolthis end of the pipettethis.end of the pipettehas been quickly thickenedand rounded in the flamethis is the finished ML43Fig. 3-5Pasteur pipettes

3 1 Making the Laboratory Readyfrom glass tubing (ML 51) and use the rubber teats(ML 43).To make a Pasteur pipette we take a piece of glass tubeabout 15 cm long and hold it in a hot flame until themiddle part is soft. We then take the tube out of theflameand pull the two ends apart. The hot soft glass is pulledthin and then cools and goes hard. We break the glasstube in the middle and put a rubber teat on each end. Inthis way we make two Pasteur pipettes. Either a Bunsenburn&r or a para& pressure stove (a ‘Primus stove’) canbe used. Make Pasteur pipettes like this, but before youmake them you must learn how to cut a glass tube andround its ends.METHODMAKING PASTEUR PIPETTES. FIGURE 3-5CUlTlNG A GLASS TUBETake a piece of glass tube and put it down flat on thebench.Take a diamond pencil and make one scratch acrossthe glass tube in the place where you want it to break.The scratch need only be quite a short one. Look atPicture A. Take the glass tube and break it in your twohands as shown in Picture B. Hold the scratch outwardsand put your two thumbs behind it. The tube will snap(break) neatly in half as shown in Picture C.If you have not got a diamond pencil, use one of thelittle metal files that are used for opening ampoules. Afile is a tool for shaping something, and an ampoule is avery small glass bottle for drugs. Any hospital dispensarycan give you an ampoule file. Look at the ampouleand ampoule file in Picture A.TUBE OUT OF THE FLAME AND PULL THE ENDSSTEADILY APART.The faster you pull the thinner will be the pipette. Theslower you pull the thicker will be the pipette. Asyou pull. the hot soft tube goes cold and hard, andyou cannot pull it out any more. By the time the tubegoes cold the thin part of it should be about 20 cmlong.As soon as the tube is cold, cut it into two Pasteurpipettes, each with about 10 cm of narrow tube.Quickly pass the end of the pipette through the flame.This will make the cut end strong and smooth. Don’thold the cut end in the flame too long or it will seal(close) up and you will have to cut or break the sealedend off.Unsealed pipettes are more often used, but it is sometimesuseful to make sterile plugged Pasteur pipettes withsealed ends. Plugged means blocked or closed. A pipetteof this kind is shown in Picture K. You will see that thethin end has been held in the flame until it is sealed. Theother end has been plugged with cotton wool which willlet air go through but stop micro-organisms getting in. Ifthe pieces of cotton wool stick out of the end of thepipette after you have plugged it, burn them off in aflame. These plugged sealed pipettes can be sterilized in apressure cooker which will kill all the micro-organismson them and inside them. The inside will remain sterilebecause one end is plugged with cotton wool and theother end is sealed. When these sterile plugged and sealedpipettes are wanted, the thin end is broken off and thethin part of the pipette is flamed. Read how to use themin Section 1.22.If you have not got a Bunsen burner, make Pasteurpipettes on a paraffin pressure stove like this.ROUNDING THE END OF A GLASS TUBEWhen a glass tube has been cut its ends are sharp andcan easily cut your fingers or spoil a rubber teat. To stopthis happening the sharp ends of a glass tube should bemade round and smooth.Hold the sharp end of the tube in the hot part of aflame, as shown in Picture D. Turn the end round in theflame a few times. When it is cool you will see its end issmooth. If you hold the end of the tube in the flame toolong it will close up completely, as in Picture E.PULLING THE PIPEIYETake a piece of glass tubing 15 cm long. Hold it inboth hands towards the top of the flame of a Bunsenburner.Keep turning the tube round in your hands. In a minuteor two the tube will become soft and start to bend,and the flame will become Yellow, as in Picture G. Waituntil the glass is really soi?.WHEN THE GLASS IS REALLY SOFT, TAKE THEMETHODSM4KING PAS ‘EUR PIPElTES ON A PARAFFIN PRESSURESTOVE, FIGURE 3-6Take off the ring on top of the stove. Take off themetal cup that spreads out the flame (the flamespreader).Hold the glass tube as shown in Picture A and make aPasteur pipette just as you would if you were making aPasteur pipette on a Bunsen burner.The ends of Pasteur pipettes often break, and it savesglass to be able to make new ends.MAKING NEW ENDS ON PASTEUR PIPETTES. FIGURE 3-5Take two Pasteur pipettes with broken ends. as inPicture B.Heat the ends. When the glass is hot and soft, pushthem together so that they stick, as shown in Picture C.Take the joined part out of the flame and heat the

Making and using Pasteur pipettes 1 3.9glass next to it as shown in Picture D. Use this soft glassto make a pipette in the usual way, as in Picture E.Make a new end on the other pipette by heating andpulling the glass at place ‘X’ in Picture E.Don’t throw away any pieces of glass tubing out ofwhich you could possibly make a Pasteur pipette.HOLDING A PASTEUR PIPEITE, FIGURE 3-6It is important to hold a Pasteur pipette properly.Hold the teat between your thumb and index finger andthe pipette itself between your middle finger and ringfinger. If you don’t know which these fingers are, look atPicture F. Your thumb comes first on your hand, yourindex finger comes second, your middle or forefingerthird. your ring finger next, and your littld finger comeslast.PRACTICE HOLDING A PASTEUR PIPETTE PROP-ERLY THE VERY FIRST TIME YOU COME TO USEIT.WASHING A PASTEUR PIPEITE. FIGURE 3-6, PICTURE GAlways keep two plastic cups on your bench, onelabelled ‘saline’. the other ‘waste’. Waste is the dirtysaline you are going to throw away. It is best to havecups of different colours. You may be given plasticbeakers instead of cups.To wash your pipette, take up some clean saline andblow it into the waste cup. Blow it out keeping the endof the pipette always ABOVE the surface of any dirtysaline there may be in the waste cup. Don’t let thepipette dip into the waste saline. Try to get all the salineout of your pipette. You will find this easier if you touchMAKING PASTEUR PIPETTESON A PARAFFIN STOVE, glass tubeMAKING A NEW END ONA PASTEUR PIPETTEB--the ring and flamespreader have beentaken off the stoveHOLDING A PASTEUR PIPETTED ‘T-he pipettes apart//little finger ’‘.SQUEEZE THE BULB BETWEENYOUR THUMB AND INDEX FINGER,HOLD THE PIPETTE BETWEEN YOURMIDDLE AND RING FINGERS keep the saline and wastecups close togetherFig. 3-6 Making Pasteur pipettes on a pressure stovelabel these cups

l-,,li .: ,) _3 1 Making the Laboratory Readyits end against the side of the cup as you blow out thelast drops of saline.Take up and blow out several pipette-fulls of saline inthis way.Keep the saline and waste cups close together. Keepyour pipette in the clean saline cup after use.ALWAYS BLOW OUT INTO THE WASTE BOWL-NOT INTO THE SALINE CUP. In this way you will keepyour saline,clean.Keep your pipette and waste cup for liquids that arenot infected, such as the washings from blood cells. Putall liquids or pipettes that may be infected. such asstools, urine, or CSF. etc.. straight into lysol. Don’t use aninfected pipette again until you have sterilized it.Different methods often need different sizes ofpipette, so make pipettes of the size that your methodneeds. Pipettes need not always be made of glass andplastic drinking straws make good pipettes for manypurposes.MAKING A STIRRING RODIt is sometimes useful to be able to stir a liquid with aglass rod. You can stir with a glass tube, but liquid getsinside the tube, and the end of the tube scratches thebottom of whatever the liquid is in. To make a stirringrod, get a glass tube and seal up both ends completelyas in Picture E, Figure 3-5. Cool the tube slowly or itmay crack, and don’t make the ends too thick. Keep thisrod for stirring and mixing.3.10 The loop (FIGURE 3-7)In the main list there are two loop-holders (ML 28) andsome nickel-chrome wire (ML 55). Take some of thewire, put it into your loop-holder and make a loop. Theseloops are used in many methods and are shown in manyfigures-look at FIGURE 1 l- 1. Loops are mostly usedfor taking a little of a specimen, such as stool or urine,and making a film on a slide. A loop is useful‘because itcan easily be flamed both before and af:er it is used. Theflame will kill any micro-organisms on the loop. A loopwhich has been flamed is therefore sterile. Flaming aninfected loop also stops micro-organisms getting on tothe bench, your fingers, or about the laboratory. If youlet organisms from specimens spread about the laboratorythey may infect you and make you ill. This isespecially important with Mycobacterium iuberculosis.One end of your loop-holder is larger than the other.This large end is the chuck (a chxk is a tool which holdssomething). You will find that part of it will unscrewthisis the collar. Unscrew the collar, and you wiil seethat the screw inside has been cut into four pieces. Thenickel-chrome wire is put into ihe middle cf these fourpieces. The screw has been drawn bigger in Picture C, sothat you can see where to put the wire.METHODMAKING A LOOP, FIGURE 3-7Take a piece of nickel-chrome wire. Bend it onceround a middle-sized nail as shown in Picture F.Take the loop off the nail, as shown in Picture G.Cut the end so as to make a loop. Use a pair of pliers.You will now have made a loop, but it will be at oneside of the piece of wire as shown in Picture H. Bend theloop so it is in the middle of the wire as in Picture I.Make sure the loop is closed.Cut the wire so that it is 6 cm long, including the loop.Unscrew the collar. Put the wire through the collara/chuck /this is a loopholder/screwML28‘landle,SOME BAD LOOPStoonot closedtyg Id+YI\too smalllie* -7 m-mcollarK this is the size and shaper\ /special wire your loop should bev V’ / iFig. 3-7The loop/A? -- chuck

Some practical details 1 3.11and into the middle of the four cuts on the chuck. Screwup the collar, and your loop is ready to use.Look at the full-size picture of a loop that has beendrawn in Picture K. Put the loop you have just made ontop of this loop. Is your loop the same size and shape?Pictures L, M, N, and 0 are some bad loops-your loopshould not be like these. Loop L is not closed up, nor isloop I, but J is closed up well. If your loop is not closedup it will not hold a drop of fluid. Loop M is too big.Loop N is too small. Loop 0 is very bad indeed.Lock at Picture C in FIGURE 3-2 to see how to flameyour loop.METHODFLAMING A LOOP, PICTURE C, FIGURE 3-2If there is something on your loop, like a big piece ofsputum or stool, try to get this off first. Dip your loopinto the edge of the disinfectant in the disinfectantbucket, or into a jar of disinfectant on the bench. Thiswill clean it. If you flame a large bit of sputum it willbubble and spit, and micro-organisms mzy Fall on to thebench without being killed.Use the top hot part of the flnma, and flame the wholeloop. There is no need to leave the loop in the flame formore than a second or two, nor need it become red hot.Many people flame the chuck quickly, but don’t leavethe chuck in the flame for more than a moment, or youwill spoil it.Help to stop harmful micro-organisms spreadingaround the laboratory. ALWAYS FLAME YOUR LOOPAFTER YOU HAVE USED IT ON AN INFECTEDSPECIMEN AND BEFORE YOU PUT IT DOWN ONTHE BENCH.Help to stop organisms from getting from one specimento another. ALWAYS FLAME YOUR LOOPBEFORE YOU PUT IT INTO ANY SPECIMEN.3.11 Some practical details (FIGURE 3-8)You have already read about some of the more importantthings in the laboratory. But there remain a few usefulthings to tell you.FOLDING A FILTER PAPER, PICTURE 8. FIGURE 3-B1 and 2. Fold the paper in half.3. Fold the paper in a quarter.4. Open out one of the folds in the paper. Put thepaper into a funnel.Filter papers are very useful in a laboratory. Waterand solutions go through them easily, but bigger thingsdo not go through and are left behind on the paper (seeSection i.5). We often filter stains before putting them ona slide, because a filter paper stops pieces of solid stainthat have not dissolved from getting on to a film andspoiling it. In Section 7.42 you will read how the proteinsin the blood are removed by filtering before theblood sugar is measured.As you read in Section 2.3, the tidiest way of labellinga bottle is with paint. But it is diRicult to paint the labelof a bottle so that the letters are straight and it looks well.You will find it helpful to draw a line on the bottle withgrease pencil first.METHODLABEkING A BOTTLE. PICTURE C. FIGURE 3-BPut the grease pencil on something firm which is atthe same height as the bottom of the letters, such as atin as shown in Picture C. Hold the pencil still and firmlyturn the bottle round. This will make a straight line onwhich you can paint. Afterwards you can easily rub outthe grease pencil line.Paint a box on the bottom of the bottle. Used like thisthe word ‘box’ means the place on a form where youwrite. Into part of the box put the data the reagent wasmade, and into the other part put your initials. Use agrease pencil so that you can rub it out when the bottleis filled up again.Careful labelling like this is very important, becauseanybody can tell exactly what reagent is in a bottle, whomade it, and when he made it.There is a useful way of making an old bottle into abeaker. The bottom of a bottle can easily be cut off andused as a cup, as shown in Picture D. Don’t heat thesebeakers made from bottles, or they will break.METHODSHOLDING A HOT TEST TUBE, PICTURE A, FIGURE 3-BIf you don’t want to burn your fingers, the easiest wayto hold a hot test tuba is to use a piece of paper. This isvary useful when doing Benedict’s test.1. Take a piece of paper.2. Fold it into a strip (a strip is something long, thinand flat).3. Fold the strip around the test tube, and pinch (hold)it between your finger and thumb close to the tube.METHODCUTTING OFF THE TOP OF A BOI-IIE, PICTURE D, FIGURE 3-BMake a small scratch on the bottle just where youwant to cut it in half. Get some long strips of blottingpaper about 25 cm wide. Wet them and wrap themround the bottle about an inch apart above and belowthe scratch.Hold the bottle above a Bunsen burner, and turn itround in the flame. As the glass gets hot between thecold wet bl&ting paper it will crack in half.

:-“.;L,. ..,’ :.’g~&~;:,, ;>/,-‘c; L:__:A i0~0iN~ A PIECE OF PAPER TOHOLD A TEST TUBEalong these lines!n--Bunsen burnerfourc PAINTING A LABEL ON A BOTTLEthese are the sizes -of the holes in rrenE A LOCALLY MADE TEST TUBE BLOCKKahnstandardtinhold the grease pencilstill and turn theand the date in thisbottle round box in grease pencilsmallCI-OSSD MAKING A BEAKER OUT OF A BOTTLEseveral thicknesses ~..__ of -.F ASLIDERACYwoodenboardhalf an inchbetween theturn the bottlethe bottle has neatI\broken in halfA/slide 2Fig. 3-8 Some practical details

Washing equipment 1 3.12Find a piece of rubber tube which is just long enoughto go round the bottle. Cut it from one end to the otherwith a pair of scissors, keeping the cut on the same sideof the tube. Put the rubber round the edge of the bottle,and you will have a useful beaker. A beaker like this canbe used for keeping slides and coverslips in before theyare washed. These beakers are shown holding pipettesin Figure 3-l 1.The test tube block shown in Picture E, FIG~JRE 3-8, iseasily made from any hard wood. It can be made withordinary carpenter’s tools, but it is best made with anelectric drill (a post drill). A- drill is something for makingholes. The block takes the three sizes of test tubesgiven in the equipment list.You will need a slide rack. Ask a carpenter to makeone from hardwood as shown in Picture F, FIGURE 3-8.Ask him to make slots (cuts) with a saw into which theslides can fit. The slots must be sloping (leaning) a little,and they must be wide enough to hold the slides. Theseracks have to be made because they cannot be bought. Ifyou cannot make a rack, stick your slides into a lump ofplasticice to hold them upright while they dry.3.12 Washing equipmentThis is very important. Even in a small laboratory it isuseful to wash most things separately.Disinfecting and washing specimen containersMany kinds of plastic or paper containers are destroyedby being autoclaved. These containers should be burnt.But polypropylene polypots (ML 14~) can be autoclavedand washed many times.If you have to wash stool containers, try to wash aslittle stool as possible. There will then be less smell whileautoclaving and the washing up water will not get sodirty. Try therefore to remove as much stool or sputumfrom a container as you can before it is autoclaved. Thebest way to do this is to put a piece of toilet paper ornewspaper in the containers before they are sent to thewards to be filled with stool or sputum. After a specimenhas been examined remove the paper from the containerwith a pair of forceps. Put it into a paper bag in akerosene tin. Later, burn the paper bag. If there is nopaper, remove as much of the specimen as you can with astick before it is autoclaved.When you have removed what stool or sputum youcan, you can either disinfect the containers in a bucket orin a pressure cooker.If you use a bucket, put all infected specimen containersinto a metal bucket of lysol (half a cupful of lysolto a bucket three-quarters full of water). Everything to bedisinfected must be covered with the lysol solution. Ifbottles or containers are to be disinfected in lysol, it mustbe able to get to every part of the inside of them. So besure to remove the cap of every bottle or container youare disinfecting in lysol. When the bucket is nearly full,put it on top of a parallln pressure stove and boil it for15 minutes. This is why the bucket must be metal andnot plastic. You can wash infected specimen containersafter they have been several hours in lysol without firstboiling them, but this is not really safe. You can also boilcontainers without first putting them in lysol. But thistoo is not really safe, because the infected water may bespilt before it has been boiled.If you use a pressure cooker, put two cupfuls of waterinto it and see that the trivet is inside it. Put infectedspecimen containers into the cooker as soon as you havefinished with them. If they smell, put the lid on. Eachmorning, or whenever the cooker is full, autoclave thecontainers to kill all the dangerous micro-organisms.There is no need to put lysol in the water, because thereis so little liquid in a pressure cooker that it cannot get tothe specimens in their containers. The harmful microorganismswill be killed by the heat of the steam, and thecontainers will be safe to wash. Five minutes at 15 lb.will be enough to kill the harmful micro-organisms. Itwill not, however, be enough to kill the spores of everymicro-organism and make the contents of the cookercompletely sterile. To do this and to sterilize syringes,etc., you must use 15 lb. for 15 minutes.When you have made infected things safe by sterilizingor autoc!aving, they will still be dirty. Wash themwith soapy water, rinse them with clean water, and letthem dry.Test tubesAlways wash them out immediately they have been used.Don’t leave dirty test tubes in a test tube rack.BloodslidesThese will be covered with oil. Put them into a detergentsolution (a teaspoonful of detergent in a pan of water).There is a special pan (ML 65) in the equipment list sothat you can do this.StoolslidesKeep these in a jar of lysol and wash them when the jar isfull.Slidesfor AAFBUnless you are keeping all positive slides for someone tocheck, break them so that they cannot be used againseeSection 11.2. If you have to use them again, washthem well, and boil them in an enamel pan in dichromatesolution. Then use them for other methods, such as thosefor stools. This solution is described below.CoverslipsPut these into a small jar or cup of lysol. Many laboratoriesthrow used coverslips away, but this is wastefuland they can easily be used several times.

3 1 Making the Laboratory ReadyCountingchambersWash these with soapy water. Be very careful of theruled area, or it may be scratched. Blot it dry with filterpaper or a soft cloth.SyringesPut these in water as soon as they have been used. Squirt(push) water through them to remove the blood. Washthem in soapy water and rinse very well to remove all thesoap.Blood pipettesWash these in water. Then, if possible, using a filterpump (see Section 3.3) suck water through them toremove the blood. Next, suck a little acetone through toremove the water. Last of all suck air through to removethe acetone.Many laboratories do not have enough pipettes to usea clean pipette on each patient, nor do they have acetoneor a filter pump. The best thing then is to rinse (wash) outa pipette with water and to use it as dry as possible byblowing out the water-see Section 7.1.If pipettes get dirty or blocked, push a thin wire downthem or use dichromate cleaning solution.Dichromate cleaning solutionChemicals for this solution are listed in Choice 11 (seeSection 13.24), and the way to make it is described inSection 3.26. It is a deep orange caustic solution and isused for cleaning any kind of glassware and some plasticequipment which cannot be cleaned in any other way. Itis probably not really necessary in either health centresor district hospitals. If you have dichromate cleaningsolution, use it like this.METHODUSING DICHROMATE CLEANING FLUIDPut pipettes in a cylinder of the solution. Put bloodpipettes in a test tube of solution. Test tubes, slides, etc.,can be put in a polythene bowl or bucket of solution. Letthe glassware stand for at least a day. Then, take it outand wash it well with water. You will find that it willnow be clean and shining.Dichromate cleaning fluid will keep for months. Use itwith the care it deserves. Only throw it away when itfinally goes green. Don’t dilute it with water. Don’t addany soap or detergent to it. Don’t keep it in a metal orenamel bucket because it may eat through the bucket.Don’t clean anything except glassware in it. Don’t putyour hands in it-use a rubber theatre glove or, betterstill, a thick post-mortem-room glove. When you finallythrow it away, pour it on to some waste ground and notdown the sink-it may spoil the drains.3.13 RoutineA daily routine is the way in which you do the things thathave to be done every day. If your laboratory is going towork well, it is very important that this routine should beright. It will be different in different laboratories, buthere is a routine for the beginning of the day that youmight find useful.If you can, find a servant to do the washing-up in theafternoon so that you can start work in the morning.When you first come to work, if you are doing all thecleaning yourself, light the pressure stove. This will heatwater to wash slides and bottles-see Section 3.12.While the water is heating, sweep the floor, then dust thebench and polish it. When this is done the water will behot enough for you to start washing. By the time youhave finished washing there will probably be enoughspecimens in the laboratory for you to start looking atthem.By having a regular daily routine like this you can besure that the ordinary things like washing and polishingare always done.3.14 Time and motion studyIn most laboratories there is much work. Very oftenthere is so much work that it is only possible to do partof it. If you are going to do as much work as you can aswell as you can, you must try to save yourself time. Thisis what ‘time and motion study’ means. It is the study ofthe time it takes to do jobs and the movements that areneeded to do them. Using time and motion study we canoften do more work with less trouble. Here are some ofthe things you can do to make your work quicker andbetter.METHODTIME AND MOTION STUDY IN LABORATORY WCAKWhen you have several tests to do, try to do themtogether, not one by one. Let us say, for example, thatyou are asked to stain films of sputum from twelvs differentpatients. First put all twelve specimens in a rowwith their labels to the front so that you can see them.Next put twelve clean slides in a row. one in front ofeach specimen. Label each slide. Then make films fromeach specimen in turn and fix them. Try to have a bigenough rack to stain them all together. By doing eachstep on all the slides together you will save time. This isnot possible with every method. Gram’s stain, forexample, must be done one slide at a time (see Section11.5).Try to make large numbers of things at a time. If youare making specimen containers, make many. If youneed much of a particular reagent, make a large volumeto save the trouble of having to make more soon afterwards.

Stains and reagents 1 3.15Keep all the things you use often as close as you canto the place where you use them. Try to walk or get upfrom your seat as little as possible.Try to make all your apparatus as easy to use as youcan. If, for example, you can get an aspirator jar to storewater, this will be easier to use than a bucket and jug(see Section 3.3).MAKINGREAGENTS3.15 Stains and reagentsMost of the reagents used for the methods in this bookare solutions of chemicals in water. If the chemical iscoloured and the solution is to be used to stain (colour)something, we call it a stain.Every reagent or stain must have in it the rightamount of each chemical. Each chemical must thereforebe weighed, and’ +he volume of the whole solution mustbe measured. Read in Chapter Five about weighing andmeasuring before you make any reagent. Weigh chemicalsin a plastic watch glass and use the Ohaus triplebeam balance (see Section 5.5). Most reagents are madein a lOO-ml stoppered measuring cylinder and are usedin polythene dropping bottles. Use the stopper to closethe cylinder while you shake it to dissolve the chemicn!.Small volumes of reagent (up to 10 ml) can be made in agraduated centrifuge tube. Hoid your thumb over the e&lof the tube when you shake to dissolve the chemical.Scrape your thumb on the edge of the tube to remove thereagent when you have finished shaking (see Pictures 8and 9, FIGURE 7-36). Make large volumes (up to 1,000ml) in a litre measuring cylinder. Except when usingstrong acids and alkalis, hold the palm (flat part) ofyour hand over the top of the cylinder when youshake.Some kinds of chemical are purer and more expensivethan others. By pure we mean that there is nothing in abottle besides that chemica!. A pure reagent contains nodirt or impurities. The purest and most expensive reagentsare often called AR reagents (analytical reagents,or special reagents for exact testing). Next come theGPR or General Purpose Reagents. Lastly come thecheapest and least pure. GPR reagents are good enoughfor all the methods in this book.Never let any chemical get from one bottle into another.Always put a clean spatula into a bottle of chemical.If you have not got a spatula, use a slide or a plasticspoon. Some chemicals spoil metal spoons, and thesespoons may spoil them. Put the right lid on the rightbottle, because a little chemical may get from one bottleto another on the lid. Keep all lids on tight, because somechemicals easily get damp (wet).Tap water or rain or well’water can be used for thereagents and methods in this book. In larger laboratoriesreagents are always made with specially pure watercalled distilled water. Distilled water is made by boilingordir-;ary water to make steam and then condensing thestean (turning it back into water again). Water madefrom steam in this way is pure. If you have distilled wateryou should use it.METHODMAKING 190 ML OF A REAGENT, FIGURE 3-91. Weigh out the amount of the chemical you need ina plastic watch glass. If your watch glass is not largeenough, use scrap paper, or plastic cups.2. Fetch a stoppered measuring cylinder (ML 18).3. Tip the chemical from the watch glass into a IOOmlstoppered cylinder. It may help to use a funnel or apiece of folded paper and a spatula. Many reagents needmare than one chemical, so you may have to weigh outseveral chemicals.4. Using water from a wash bottle, wash the chemicalon the watch glass through the funnel and into the cylinder.5. Fill the cylinder up to the IOO-ml mark. This iscalled ‘making it up to 100 ml’. You can, if you wish, fillit to about 90 ml from a tap and then exactly to the markwith a wash bottle or a Pasteur pipette. Look at thecylinder from the side when getting the top of the solution(the meniscus) to the IOO-ml mark (see Picture V,Figure 5-3).6. Put back the stopper and shake the cylinder todissolve the chemical.7. When the chemical is all dissolved, pour the reagentinto a bottle. This will often be a polythene droppingbottle, which holds about 100 ml.8. Label the bottle and write your initials and the datein the box at the bottom.There are many reagents to be made up in this way.They are not all exactly the same, and you will haveto change what you do slightly in the methods thatfollow.METHODS3.16a MAKING 3% ACID ALCOhOL-250 MLMeasure 7.5 ml of concentrated (strong) hydrochRoricacid into a graduated centrifuge tube-you will find aPasteur pipette useful. Don’t use a graduated pipette, becauseyou may get acid in your mouth. It is dangerous.Tip the acid into a l,OOO-ml cylinder. Add spiritto .250 ml. Shake with the palm of your hand over thetop of the cyl:nder. Pour this mixture into a 260-ml washbottle.You can also make 3 ml of hydrochloric acid up to100 ml with spirit and keep the mixture in a droppingbottle. But you will probably use enough of this reagentto make it worth making up 250 ml at a time.3% acid alcohol is used for the Ziehl-Neelsen methodfor staining sputum for Mycobacterium tuberculosis.Look at Section 11 .l .

.-:-3 1 Making the Laboratory Ready/'ML38 /spatulaMlthe chemicalwatch glass\,stopperedmeasuringcvlinder‘&ML 52if there is too muchchemical to go in awatch glass, use aplastic cup or asheet of paper!-Y&funnelcylinderat once iflterthe remains of thechemical in the wattglass are being washtinto the bottleI4 ---_label the bottlelook at themeniscus fithis sidepalm of your handover it whenname and the dateFig. 3-9 Making a reagent3.16b MAKING 1% ACID ALCOHOL-260 ‘MLMake this in exactly the same way as 3% acid alcoholdescribed above, but make 2.5 ml of hydrochloric acidup to 250 ml with spirit, or 1 ml of acid up to 100 mlwith spirit.1% acid alcohol is used for the Ziehl-Neelsen methodfor staining skin smears for Mycobacterium leprae as inSection 11 .l 1 b. 3% acid alcohol can be used for Mycobacteriumleprae, but 1% acid alcohol is better.3.17 MAKING 10% BARIUM CHLORIDE-100 MLWeigh 10 g of barium chloride into a 100-ml measuringcylinder. Make it up to 100 ml with water. Shake todissolve and keep the solution in a polythane droppingbottle.This reagent is used for testing for bilirubin byFouchet’s method--see Section 8.8.3.?6 MAKING BENEDICT’S REAGENT-l.000 Ml.Weigh 17.3 g of copper sulphate. Copper sulphateforms deep blue crystals. Put it in a 100-ml measuringcylinder. Add about 100 ml of water. The exact volumeof water does not matter. Shake the cylinder to dissolvethe copper sulphate.While the copper sulphate is dissolving, weigh 173 gof sodium citrate and put it into a l,lXlO-ml cylinder.Weigh 100 g of sodium carbonate. Add this to the1 .OOO-ml cylinder. There is too much sodium citrate andtoo much sodium carbonate to weigh on a watch glass.So, if you are using the Ohaus balance, weigh thesechemicals in a plastic cup.Fill the l,OOO-ml cylinder to the 600-ml mark withwater and shake it to dissolve the sodium carbonate andthe sodium citrate. When the sodium carbonate and thesodium citrate have all dissolved, add the copper sulphatesolution in the loo-ml cylinder a little at a time.Shake the 1,600-ml cylinder each time you add somecopper sulphate solution. The mixture may go cloudy asyou add the copper sulphate solution. But it will go clearagain as you shake.When you have added all the copper sulphate solution,there will be about 700 ml of liquid in the 1 ,OOO-mlcylinder. Fill the cylinder to the l,OOO-ml mark withwater. Mix well. Keep some of the Benedict’s solution in

Buffers 1 3.20aa wash bottle and the rest in a IBOO-ml polythene reagentbottle (ML 6).This reagent is used for testing the urine for sugarseeSection 8.3.3.19 MAKING BRILLIANT CRESYL BLUE SOLUTION-250 MLWeigh out O-2 g of brilliant cresyl blue and dissolve itin 100 ml of saline. Keep it in a polythene droppingbottle. This will la? a very long time, but it is not easy tomake up a smaller volume, as it is not possible to weighout less than O-2 g with the Ohaus balance.Cresyl blue solution is used for staining reticulocytes--seeSection 7.23.3.20a BuffersRead about btiers in Section 1.7, about gastric washingsin Section 11.4, and about the various kinds ofphosphate in Section 2.4. The list of chemicals containstwo kinds of potassium phosphate. These are potassiumdihydrogen phosphate (KH,PO.J and dipotassiumhydrogen phosphate (K,HPO,). If you have not got thesephosphates you can use sodium phosphate for two ofthe three buffers you will need. On most bottles ofchemicals you will see a number after the name and theformula, such as, for example, potassium dihydrogenphosphate, KH,PO,, 136.09. This number is the molecularweight, and it may help you to recognize the rightphosphate.The buffer for gastric washings is best made intoa solution, but the buffer for Leishman’s stain is bestmade as a powder by mixing two of the phosphates.When you make buffer powder for Leishman’s methodtake any one phosphate from the list P- idf acid saltsand any one other phosphate from the list B ofalkaline salts. You will probably use 13.9 g ofKH2P0, and 17.2 g of K,HPO,. The buffer for gastricwashings only requires any one of the alkaline saltsfrom list B.METHODS3.20b MAKING THE BUFFER FOR GASTRIC WASHINGS-50 MLChoose one of the alkaline phosphate salts from list 8in the section above, and weigh it into a measuringcylinder. Add water to 50 ml. Shake hard-the waterwill get cold as the phosphate dissolves. Put O-5 ml ofthis solution into each of several universal containers.Sterilize these in the pressure cooker (ML 691 with theirlids on loosely. As soon as the bottles come out of thecooker, screw their lids down tight. They will be sterile-that is all the micro-organisms inside them will havebeen killed. Don’t open them until you want to usethem, or micro-organisms may get in and contaminatethem (make them unsterile again). If you have any solutionleft over, put it in a glass bottle with a metal screwcap and sterilize it in the cooker. You can then fill andsterilize other universal containers when you want them.This buffer is used for adding to gastric washingswhen they are to be cultured for Myco. tuberculosisseeSection 11.4a.3.21a MAKING THE BUFFER POWDER FOR LEISHMAN’SMETHOD-ABOUT 30 9Choose one of the acid phosphates from list A inSection 3.20a. Choose also one of the alkaline phosphatesalts from list 8. Look to see if they are in very finepowders. If they are you can weigh them as they are.But, if they are lumpy, you will first have to grind theminto a powder. KH,PO, is usually a fine powder, butK,HPO, is often lumpy. The best way to grind a lumpychemical into a fine powder is to use a clean pestle andmortar; so get one if you can. Dispensaries usually havethem. If you have not got a pestle and mortar, tip someof the chemical on to a clean newspaper and roll andcrush it with a clean bottle. Lumpy K,HPOI can easily becrushed into a fine powder this way.Weigh each of your two finely powdered chemicalsinto a clean wide-mouthed jar, ML 62. Mix and shakethem well. Label the jar ‘Leishman buffer powder’.For use, add a little on the end of a spatula. aboutA. Take the weight shown of any one of these acid salts:Potassium dihydrogen phosphate anhydrous, KH,PO, 136.09Sodium dihydrogen phosphate anhydrous, NaH,PO, 119.98Sodium dihydrogen phosphate hydrated, NaH,PO,.2H,O 156.01For making 100 mlof buffer forgastric washingsnot needednot needednot neededFor makingLeishman’sbuffer powder13.9 g12.2 g15.9 gB. Also take the weight shown of any one of these alkaline salts:Dipotassium hydrogen phosphate anhydrous, K,HPO, 174.18Disodium hydrogen phosphate anhydrous, Na,HPO, 141.97-Disodium hydrogen phosphate hydrated, Na,HP0,.2H,O 178.0 (alsocalled Sorenson’s salt)Disodium hydrogen phosphate hydrated, Na,HPO,. 12H,O 358.1624.5 g20.0 g17.2g13.9 g25.0 g 17.6 g52.0 g 35.5 g

,“.’ -_3 1 Making the Laboratory Ready250 mg, to each 100-ml ‘polystop’ bottle of water you useto make Leishman buffer. If you were not to use finelypowdered phosphates, your 250 mg might be a lumpof one kind of phosphate only.This buffer is used for Leishman’s method for stainingthin blood films-see Section 7.11.3.21b MAKING BUFFER FOR THE SOLUBILITY METHOD FORHAEMDGLOBINS A AND S-100 MLCarefully weigh out exactly 13.5 g of potassium dihydrogenphosphate (anhydrous KH,PO,). Put most of itinto a loo-ml measuring cylinder with a spatula andwash the rest in with a wash bottle of water through afunnel (see Figure 3-9, Picture 4).With equal care weigh out exactly 23.7 g of dipotassiumhydrogen phosphate (anhydrous K,HPO,J and addthis to the cylinder in the same way.Then without using a funnel add 1 g of white saponina little at a time. Shake each time you add some. Youwill get a soapy frothy solution. Brown saponin does notwork.Wait until most of the froth has settled. Make up thevolume with water to exactly 100 ml, and again shakehard to dissolve. Keep the buffer in a lOO-ml droppingbottle with a cap on the spout. Keep it in a refrigerator ifpossible. Make up fresh working solution each day, asdescribed in Section 7.26.For this method the phosphates must be anhydrousthatis they must be dry, so keep the lids of the bottlesscrewed on tight.3.22a MAKING ‘CELLOPHANE’ COVERSLIPS FOR THE THICKSTOOL SMEARMeasure 50 ml of glycerol into a screw-capped [. r(ML 62). Add 30 ml of water, .lO ml of the malachnegreen solution described in Section 3.34, and 10 ml offormalin solution (not form01 saline).Get the ‘Cellophane’ sheet described as ML 148aunder Choice 16 in Section 13.29. Put several sheetstogether and cut them into strips just narrower than a&de (22 mm), using a slide as a guide. Then cut thestrips into pieces about 30 mm long. The cellophanesneets easily slide over one another as they are cut together,so, if you have a stapling machine, staple thorntogether while cutting. This ‘Cellophane’ sheeting maybe sold in ro?ls 22 mm wide, which will make your jobmuch easier.Soak the coverslips you have made in the malachitegreen solution for at least a day before they are used.These coverslips are used in the ‘Cellophane’ tnicksmear method in Section 10.2b. Both the malachitegreen and the formalin can be left out if necessary. Theformalin has been put in as an antiseptic to make theslides safer to handle.Carbol fuchsin is used for staining the sputum forMycobacierium tuberculosis by the Ziehl-Neelsenmethod. It is also used for staining skin smears forMycobacterium leprae. Two ways of making up strongcarbol fuchsin are described. One is for the older ‘hotmethod’. The other is for the newer ‘cold method’. Theword ‘hot’ refers to the way it is used, not the way it ismade. Make up the reagents for only one of thesemethods. It is probably best to choose the hot one.METHODS3.22b MAKING CARBOL FUCHSIN FOR THE HOT ZIEHL-NEELSEN METHOD-ABOUT 675 MLFind a tin which will hold at least 600 ml. Put it on thepan of an Ohaus balance. Balance it with the tareweight. Set the weights to 25 g. Add phenol until thepointer is at ‘0’ again. Set the weights to 30 g. Add basicfuchsin until the pointer is at ‘0’ again. You will nowhave a mixture of 25 g of phenol and 5 g of basic fuchsinin the tin. Heat the mixture slow/y over a flame until thephenol is melted (has become liquid) and the basic fuchsinhas dissolved. All the fuchsin need not dissolve, butmost of it should dissolve. Stir the heated mixture with aspatula. A little ‘steam’ may come off the mixture, but itmust not boil. Let the mixture cool. Add 50 ml of spiritand 500 ml of water. A greenish yellow scum (a scum isanything on the surface of a liquid) will come on thesurface of the liquid in the tin. Pour this liquid into a litrepolythene reagent bottle. Label it ‘Strong CarbolFuchsin-hot method’. Filter some of it through a filterpaper and a funnel into a polythene dropping bottle.Stain your slides with stain from this dropping bottle.When the dropping bottle is empty, refill it from thel,OOO-ml plastic reagent bottle, filtering Phe stainthrough filter paper and a funnel as you do so.Keep the tin and the funnel for carbol fuchsinonly. Don’t try to wash them: they are very difficult toclean.3.23 MAKING STRONG CARBOL FUCHSIN FOR THE COLDZIEHL-NEELSEN METHOD-600 MLMake this stain in the same way as tor the hotmethod. But use 50 g of phenol, 25 g of basic fuchsin,25 ml of spirit, and 500 ml of water. Last of all add 15drops of ‘Teepol’. Teepol is a detergent. If you have notgot any Teepol (or any Tween 60, which is anotherdetergent), use whatever detergent you haveit willprobably work. Some people do not use any detergentat all.3.24 MAKING DILUTE CARBOL FUCHSIN- MLMeasure out 6 ml of strong carbol fuchsin (eitherkind) in a graduated centrifuge tube. Pour it into a polythenedropping bottle. Add 94 ml of water to the droppingbottle.Dilute carbo! fuchsin is used for Gram’s stain (seeSection 11.5).

Buffers 1 3.253.25 MAKING CRYSTAL VIOLET STAIN-1W MLWeigh out 0.5 g of crystal violet. Tip it into a 199mlmeasuring cylinder and make it up to 199 ml. Shake todissolve and pour it into a polythene dropping bottle.Crystal violet stain is used for Gram’s method (seeSection 11.5).Formaldehyde, B.P.‘. Add to it 8.5 g of sodium chloride.Sodium chloride is the ordinary salt that you put on yourfood. If you have no GPR sodium chloride, use any kindof ordinary salt. Make up to 1,000 ml with water. Shaketo dissolve. Store in a polythene reagent bottle.Formol saline is used far preserving specimens forhistology (see Section 4.10).3.26 MAKING DICHROMATE CLEANING FLUID-l,000 Ml.Fill a litre polythene measuring cylinder to the 909mlmark with water. SLOWLYadd ID9 ml of concentratedsulphuric acid. Use ‘commercial’ or ‘technical’ impuresulphuric acid and not the more expensive pure kind.Add the acidslowly, a little at a time, and keep your eyesaway from the top of the cylinder. There will be a noiseas you add the acid and the mixture will get hot. Add20 g of ‘commercial’ or ‘technical’ potassium dichromate.Make this into a fine powder before you add itand it will dissolve more easily. If necestary stir themixture with a long glass tube. Pour most of it into anungraduated polythene measuring cylinder, and keepsome of it in a test tube. It i6 dangerous-so be careful IDichromate cleaning fluid is used for cleaning glassware(see Section 3.12).3.27 MAKING EHRLICH’S REAGENT-250 MLWeight out 0.7 g of para-dimethyl-aminobenzaldehydeand tip it into a l,OOO-ml polythene reagentbottle. You will probably find it easier to tip itthrough a funnel as in Picture 3, Figure 3-9. Pour intothe bottle 150 ml of concentrated hydrochloric acid and190 ml of water. Shake to dissolve. Pour some into apolythene dropping bottle for daily use.Ehrlich’s reagent is used for testing urine for urobilinogen(see Section 8.8).3.28 MAKING FIELD’S STAIN A OR 8-100 MLWeigh out 2.5 g of the blue Field’s stain A. Tip it intoa loo-ml measuring cylinder and make it up to 199 mlwith water. Shake to dissolve and filter the stain into a120-ml wide-mouthed jar with a screw cap (ML 52).After some days or weeks a scum will form on the topof the stain. This will spoil the stain, so you must filter itregularly-say every Monday morning. Keep the bottlefiled up with stain, or you will not be able to reach itwith your thick blood film. Throw away the stain once amonth and make new stain. If you stain many films,throw it away more often than this.Make up Field’s stain 8 in exactly the same way asyou make up Field’s stain A.Field’s stains A and B are used for staining thick bloodfilms (see Section 7.31).3.29 MAKING FORMOL SALINE-1,000 MLFill a litre cylinder to the loo-ml mark with ‘Formaldehydesolution’ or, as it is sometimes called, ‘Liquor3.30 MAKING FOUCHET’S REAGENT-100 MLWeight out 1 g of ferric chloride and tip it into agraduated centrifuge tube. Fill the centrifuge tube to thelo-ml mark with water. Shake to dissolve. While it isdissolving, weigh out 25 g of trichloracetic acid and tip itinto a loo-ml cylinder. Fill the cylinder to the 50-mlmark with water. Shake to dissolve.Tip the ferric chloride solution into the trichloraceticacid solution. Mix and make up to the 1 OO-ml mark withwater. The mixture is Fouchet’s reagent. Keep it in apolythene dropping bottle that lets the reagent comeout slowly drop by drop.Fouchet’s reagent is used for testing the urine forbilirubin (see Section 8.8).3.31a MAKING HAEMOGLOBIN DILUTING FLUID-l.000 ML1. USING SODIUM CARBONATEWeigh out ‘IO g of sodium carbonate. Tip it into a litremeasuring cylinder. Make up to the 1 ,OOO-ml mark withwater. Mix well. If you are using the Lovibond comparator,pour some of the solution into a polythene washbottle. Keep the rest in a litre polythene reagent bottle.Choose a wash bottle with a small hole in its tube. Thiswill make it easier to fill a Lovibond cell to the lo-mlmark exactly. If you are using the Grey wedge photometeror the EEL calorimeter, keep all the solution in alitre polythene reagent bottle. Using a lo-ml pipette,measure several lo-ml volumes into universal containers.Use these for the tests.If you have ammonia in your laboratory, always usethe ammonia solution described below instead of thesodium carbonate solution. Ammonia is included asChoice 9, Section 13.22.2. USING AMMONIAFill up a litre cylinder to the l,OOO-ml mark withwater. Add O-4 ml of concentrated ammonia. This is alsocalled SG O-880 or SG O-910 ammonia. Measure thiswith a Pasteur pipette in a graduated centrifuge tube.Strong ammonia is dangerous. Don’t pipette it bymouth, because it may make you cough. Put theammonia solution in the same kind of bottle, and use itin the same way as you would the sodium carbonatesolution. You can also make up this solution by addingfive drops of concentrated ammonia to 250 ml of waterin a wash bottle. This may be easier.This highly alkaline solution does not keep well. Intime it takes up an acid gas called carbon dioxide from

.::. .,. _,\3 1 Making the Laboratory Readythe air and comes to have a more neutral pH. Suchsolutions may not work, so don’t keep ammonia solutionsfor more than a week.3.31b MAKING DRABKIN’S SOLUTION-1,000 MLDrabkin’s solution is the diluting fluid for the cyanmethaemoglobinmethod for measuring haemoglobin. Itcan be made either from special tablets (see ML 127(b),Section 13.20) or from potassium cyanide and potassiumferricyanide. Drabkin’s solution is very dilute, andvery small quantities of these chemicals are needed. Ifyou are careful you can weigh them on the Dhausbalance.Weigh 200 mg (one-fifth of a gram) of potassium ferricyanideand 500 mg (half a gram) of potassium cyanide,and make up to 1,000 ml with water.3.32 MAKING LUGOL’S IODINE-100 MLWeigh out 1 g of iodine. Tip it into a loo-ml measuringcylinder. Weigh out 2 g of potassium iodide. Tip thisalso into the measuring cylinder. Mk them well. Add alittle water and mix again. Fill the measuring cylinder tothe lOO-ml mark with water. Shake to dissolve. Keepthe solution in a polystop bottle.Lugol’s iodine is used for Gram’s stain (see Section11.5). Iodine spoils metal, so don’t let it touch metal,and don’t get it on the balance.3.33 MAKING LEISHMAN’S STAIN-100 MLTake a polystop bottle and fill it with 100 ml of water.Make a mark on it where 100 ml comes. Take a secondclean DRY polystop bottle, and make a mark on it at thesame height as on the first bottle. Weigh out O-2 g ofpowdered Leishman’s stain and add it to the secondbottle. Fill it to the mark you have made with methylalcohol. You will have added 100 ml and will not havewet and spoilt the methyl alcohol by measuring it in awet measuring cylinder.Put the stopper on the bottle and shake hard. Shakeseveral times during the rest of the day and leave thebottle overnight. It will be ready to use in the morning.Never let the smallest amount of water or buffer getinto the stain. If you do the white cells will not be fixedand will not stain. Even if you measure methyl alcohol ina wet cylinder. it will be spoilt. This is why we have toldyou to measure methyl alcohol straight out of the stockbottle to a measured mark on a polystop bottle.When you need to make more stain, add morepowdered Leishman’s stain and more methyl alcohol tothe same lOO-ml mark on the bottle.Leishman’s stain usually works better as it matures(gets older). It is thus useful to make some stain and letit mature in another tightly stoppered bottle before youuse it.Methyl alcohol evaporates much quicker than water,so keep the bottle tightly stoppered.Leishman’s stain is used for staining blood films (seeSection 7.12).3.34 MAKING MALACHITE GREEN SOLUTION-100 MLWeigh O-3 g of malachite green. Tip it into a 100 mlmeasuring cylinder. Make up to the 188 ml mark withwater. Shake to dissolve and keep the stain in a polythenedropping bottle.Many people like using malachite green instead ofmethylene blue for the Ziehl-Neelsen method (seeSection 11.1).3.36 MAKING METHYLENE BLUE IN ACID ALCOHOL-500 ML.A. 8% ACID FOR MYCO. TUBERCULOSISTake a 1 .OOO-ml measuring cylinder. Fill it to the 170-ml mark with spirit.WITH GREAT CARE AND VERY SLOWLY fill thecylinder to the 210-ml mark with concentrated sulphuricacid. You will be adding 40 ml of acid. Add the acid alittle a? a time. The mixture will get hot and spit. But,because it is at the bottom of a big cylinder, it will notspit much if you only add the acid slowly.Let the mixture cool.Fill the cylinder to the 500-ml mark with water. Whilethe mixture is cooling, weigh out 3 g of methylene blue.Tip this into a litre reagent bottle. Pour the mixture ofacid alcohol and water into the bottle. Shake to dissolvethe methylene blue. Use the methylene blue acid alcoholin a polythene wash bottle. Malachite green cannot beused instead of methylene blue for this method.8. 1% ACID FOR MYCO. LEPRAEMake up this weaker acid for Myco. ieprae in thesame way that is described above except that 5 ml ofconcentrated sulphuric acid are added and not 40 ml.Label your bottles ‘8% acid-alcohol methylene bluefor Myco. tuberculosis’ and ‘1% acid-alcohol methyleneblue for Myco. leprae’. These solutions are for the coldZiehl-Neelsen method (see Section 11 .l 1.3.36 MAKING OCCULT BLOOD REAGENTWeigh out 1 g of ortho-tolidine (also written o-tolidine). 0-toluidine will not work. Tip it into a universalcontainer. Weigh out 8 g of barium peroxide. Tip thisinto the universal container. Put the lid on the containerand shake it well to mix these two dry powders.Find a cork which fits the container as in Picture X,Figure 10-13. Using a pair of strong scissors or tinshears, cut a strip of tin about two inches long and halfan inch wide. Cut points at both ends as shown in thispicture. Bend the strip down the middle and stick oneend into the cork. The other end makes a useful smallspoon or spatula.You will want 200 mg of powder each time you make

Buffers 1 3.37/fill the bottle with {I- -:,!water :--,_-‘. __.I-_If’ I’---~~ - ‘:’ !OL_~C _ __.i-shake hardthis is thebottle ofphenolif the phenolis liquid youwill have topour itphenol at least an inchdeep on the bottom ofthe bottle‘liquidrefill the stockbottle, shake itand leave it untilphenolliquid phenol ’I blML5 label the bottleleave the bottle untilthe following morning;y-p.. 5 . . ..‘.y-:,r 1;. . ‘:. . .tphenol saturated waterFig. 3- 10 Making Pandy’s reagentup the reagent. Take up what you think is 200 mg andweigh it. Are you right-is it 200 mg? If it is not about200 mg. try again. You will soon be able to guess 200mg quite well. It is not easy to weigh 200 mg on theOhaus balance, but it can be done accurately enough.Make sure the beam is exactly at ‘0’ when you start andwhen you finish.This reagent is used for testing for occult blood in thestoolssee Section 10.10. Ortho-tolidine is dangerous,so wash your hands after you have used it and don’t spillit around the laboratory.5. Carefully pour some of the watery layer at the topof the stock bottle into a polythene dropping bottle.Don’t pour off the layer of liquid phenol at the bottom ofthe stock bottle. Don’t shake the bottle before you pouroff the reagent. Label the watery layer you have pouredoff ‘Pandy’s reagent’.When the watery layer in the stock bottle is nearlyfinished, add more water, shake it, and leave it until thenext day.Pandy’s reagent is an easy way of measuring the CSFprotein-see Section 9.10.3.37 MAKING PANDY’S REAGENT, FIGURE 3-101. Put some phenol into a 1 .OOO-ml polythene bottle.Make a layer about an inch deep on the bottom of thebottle.2. Fill the bottle half full with water.3. Put the cap on the bottle and shake the mixturehard for about 5 minutes. The mixture will become turbid(cloudy, milky). Leave the bottle until the next day.4. You will see a layer of liquid phenol at the bottomand a clear watery layer at the top. This watery layer is asaturated solution of phenol in water and is Pandy’sreagent.3.38 MAKING PAS TEST STRIPSCut filter paper or blotting paper into strips about1 cm wide and about 5 cm long.Weigh 1 g of ferric chloride and dissolve it in 25 ml ofwater in a loo-ml measuring cylinder.Soak the filter paper strips in the ferric chloride solution-theywill go bright yellow. Keep the unused ferricchloride solution in a polythene dropping bottle until youneed it again. Leave the strips to dry in the sun. Keep thedry strips in a box or tin until you want them.These strips are used for testing the urine for a drugcalled PAS (see Section 8.91.

_‘ ).,,3 1 Making the Laboratory Ready3.39 MAKING ROTHERA’S REAGENTA. STANDARD ROTHERA’S REAGENTPut a few crystals of sodium nitroprusside on a sheetof clean newspaper and crush them to a fine powder byrolling them with a clean dry glass bottle. Weigh outabout half a gram (0.5 g) of the fine powder and mix itwell with about 100 g of ammonium sulphate. Store thepale pink powder you get in a screw-capped jar (ML 62).The powder goes dark blue after a few weeks and maynot work so well, so do not make up too much of thereagent at a time.Rothera’s reagent is used for testing the urine foracetone--see Section 8.7.B. MODIFIED ROTHERA’S REAGENTWeigh 3 g of sodium nitroprusside into a plastic cup.Grind it into a fine powder with the bottom of a testtube. Add 100 g of ammonium sulphate and 50 g ofanhydrous sodium carbonate. Mix well and keep themixture in a wide-mouthed screw-capped jar.Modified Rothera’s reagent is used for testing theurine for acetone+(see Section 8.7).3.40 MAKING SALINE-l.090 MLWeigh 8.5 g of sodium chloride and tip it into a litremeasuring cylinder. Add water to 1.008ml mark. Shaketo dissolve. Pour the saline solution into a litre reagentbottle.Saline is mostly used for blood grouping and crossmatching (see Section 12.4) and for examining thestools (see Section 10.2). Keep some saline in a washbottle, some in a polystop bottle, and some in a cup witha Pasteur pipette in it-see Figures 3-6 and 3-l 1.3.41 MAKING SATURATED SODIUM ACETATE SOLIJTION-ABOUT 100 MLFill a polythene dropping bottle with sodium acetate.Then fill it with water. Shake hard. About half thesodium acetate will dissolve in the water and give you asaturated solution of sodium acetate on top of a layer ofcrystals. Pour off the saturated solution as you need it.When the bottle empties, add more water and shakeagain. Remember, the solution will only be saturated aslong as there is a layer of solid sodium acetate at thebottom of the bottle.This reagent is used for testing the urine for urobilinogen(see Section 8.8).3.42a MAKING 3.8% SODIUM CITRATE SOLUTION-50 MLWeigh out very carefully 1.9 g of sodium citrate. Tip itinto a loo-ml cylinder. Add water up to the 50-ml mark.Shake to dissolve. Measure O&ml volumes into severalbijou bottles.Micro-organisms easily grow in sodium citrate solu-tion; 80 Only make up a little at a time, and don’t keep ittoo long. Keep it in a refrigerator if you have one. Youcan also sterilize the bijou bottles of sodium citrate solutionin a pressure cooker, just as you do with the bufferfor gastric washing8 (see Section 3.20). Sterilizing thesolution will kill the micro-organisms in it so that it willkeep for ever.3.8% sodium citrate solution is used for theWestergren ESR (see Section 7.391.3.42b MAKING 20% SODIUM HYDROXIDE-100 MLWeigh out 20 g of sodium hydroxide into a IOO-mlmeasuring cylinder. Add water to the loo-ml mark. Letthe sodium hydroxide dissolve and pour it into a polythenedropping bottle. Keep the cap on the droppingbottle and on the stock bottle of sodium hydroxide pellets.Sodium hydroxide easily absorbs water from the airand becomes wet.This solution is used for examining skin scrapings forfungi (see Section 11 .15). Sodium hydroxide is a strongalkali, so handle it with care.3.43a MAKING SULPHONE TEST PAPERSWeigh 2 g of para-dimethyl-amino-benzaldehyde intoa loo-ml measuring cylinder. Fill it to 15-ml mark withhydrochloric acid. Mix and then fill it to the loo-ml markwith distilled water.Pour this mixture into a IOO-ml bottle with a cap andlabel it ‘Strong reagent for Suiphone test papers’.When you want to make some test papers, put 2 ml ofthis strong reagent in the bottom of a measuring cylinder.Fill to the loo-ml mark with water. Wet Some1 l-cm filter papers (ML 22bI with this solution, let theexcess solution drop off, and let them dry. Keepthe papers in a tin with a lid away from light until youwant to use them.These papers are used for testing the urine for sulphones(see Section 8.10aI.3.43b MAKING 3% SUCPHOSALICYLIC ACID-100 MLWeigh 3 g of sulphosalicylic acid. Sulphosalicylicacid is the same as salicylsulphonic acid. Tip it into alOO-ml measuring cylinder. Add water to’ the 7 OO-mlmark. Shake to dissolve. Keep the solution in a polythenedropping bottle.This 3% solution is used for measuring the CSF protein(see Section 9.13).3.44 MAKING 20% SULPHOSALICYLIC ACID-100 MLWeigh 20 g of sulphosalicylic acid. Tip it into a lOOmlmeasuring cylinder. Add water to 100 ml. Shake todissolve. Keep the solution in a polythene droppingbottle.20% suiphosalicylic acid is used for testing the urinefor protein (see Section 8.31.

A description of Figure 3-11 1 3.463.45 MAKING WHITE CELL DILUTING FLUID-100 MLAdd 2 ml of glacial acetic acid or 1 ml of concentratedhydrochloric acid to 166 ml of water. Add two or threedrops of crystal violet stain to colour the fluid a paleviolet. Keep the fluid in any bottle. Some people like tokeep it in a bottle with a 1 ml pipette going through thecork.This fluid is used for counting white cells (see Section7.301.A HEALTH CENTRE LABORATORYDESCRIBED3.46 A description of Figure 3-l 1Let us imagine that FIGURE 3-l 1 is a picture of yourlaboratory and that you work in a health centre. Thelaboratory is on a long bench, and it could be part of aroom that is also used for something else. It has beendrawn very carefully and shows most of the things describedin the main equipment list. You will probably notkeep all the equipment in exactly the same places inwhich it is shown here. For example, it might be best tokeep many of the smaller things in the drawer (1). Gothrough this section and Figure 3-1 I very carefully to seeifyou have all the things you need. If you have not gotthem ALL, try very hard indeed to get them. DoEVERYTIIIb4G you read in this section to get yourlaboratory ready.Health centres are needed in very large numbers, sothey have to be made for little money. You will see thatthe health centre in FIGURE 3- 11 has a tin roof and noceiling. Its walls (2) are made of whitewashed mud andpoles (sticks). The mud has been mixed with an insecticide(a chemical to kill insects) to kill the termites (whiteants). There is no glass in the windows, and the shutters(3) are made of wood.There is a window in the right-hand wall of the laboratory.This is so that light from a bright sky can be usedfor the microscope. Many laboratories will have to usethis kind of light for their microscopes. In this laboratorya car battery is also used to light the microscope lamp.A long bench (4) runs along one wall. Above thebench are three shelves (5, 6, 7). There is no runningwater, so water is brought from a well in a bucket (8).This water is for the general use of the laboratory and isalso for washing hands. For this there is soap (9) and atowel (10). Because there is no running water, cleanwater is taken out of one bucket with the jug (11) andpoured over the hands into a waste bucket (12). Threemore buckets are needed. One bucket (13) holds a disinfectantsolution (see Section 1.19), such as lysol, and isfor infected specimen bottles and slides. Half a cup oflysol has been added to half a bucket of water. The capsof specimen bottles are unscrewed and care is taken tosee that the bottle (or whatever else is infected) is coveredby the disinfectant solution. To kill the harmful microorganismsthe infected bottles and slides must stay in thebucket under the disinfectant solution for at least one daybefore they are washed. Bucket 14 is empty and is forthings to be washed which are not infected, such as bloodslides. Some people like to have a bottle for infectedslides on the bench. Bucket 15 is for things to be burntsuch as cotton wool swabs, etc. Notice that all thesebuckets are labelled so that they can be used the rightway.Infected coverslips are kept separate from infectedslides when they are washed. When a saline smear ofstool has been looked at, the coverslip is pushed off theslide with a loop into the cup of lysol (16). The coverslipsare then washed separately from the slides. If theslides are difficult to wash they should be boiled in adetergent solution in the pan (112) (ML 65).Because there is no sink, slides are stained across abucket (18) (see Section 3.2). The slides rest on two glasstubes ( 17). There is a pair of forceps ( 19) hanging on anail. These are used to pick up stained slides so that staindoes not get on the fingers. A test tube brush (20) hangsnear this bucket.The stains are on a shelf on the left-hand wall. Thereare ‘Polystop’ bottles with Leishman’s stain (21) andLeishman buffer (22). There are also wash bottles ofwater (23), saline, Benedict’s reagent (24), and 3% acidalcohol (25). Next come several reagents in droppingbottles, dilute carbol fuchsin (26), crystal violet (27),and malachite green (29).On the bench there is a slide drying rack (30), a reportbook (3 1) and several specimens (32). These specimensare on top of the patients’ treatment cards. Near thespecimens is a rubber stamp pad (33), and on the edge ofthe shelf just above it are some rubber stamps (34).These are held between two nails hammered into thefront of the shelf. On the front edge of the bench is a handcentrifuge. On the back of the bench near the middleis the equipment for Field’s stain. There is Field’sstain A (35), a cup of water (36), Field’s stain B (3 7).Near by there is a tripod (38), a square of gauze (39),and a Bunsen burner (40). The Bunsen burner is joinedby a rubber tube (4 1) to the reducing valve on a cylinderof ‘Afrigas’ (42). The rubber tube goes through a hole onthe bench. There is a spare cylinder of ‘Afrigas’ underthe bench (43). Both cylinders of gas should, if possible,be outside the room. If the gas leaks inside the roomthere may be an explosion (see Section 3.4). The ‘on-off’key for the ‘Afrigas’ is hanging on a nail on the bottomshelf where it is easily found (44). The spanner forchanging the ‘Afrigas’ cylinder is hanging up out of theway (45).In the middle of the bench are two cups: one is forsaline (46) and one is for waste (47). There is a Pasteurpipette in one of them. These cups are mostly for bloodtransfusion methods, but they are also useful in healthcentres (see Picture G, FIGURE 3-6).Behind the cups of saline is a pressure cooker (48).Many people prefer to put all infected things into thisrather than into the bucket of lysol disinfectant (14).Next come two test tube blocks (49 and 50) of thelocaily made kind shown in Picture E, FIGURE 3-8. In

J”, “; -;’ . - . ’ (I/.,“..’ .‘,,,/ :‘. ‘., .,; ,,, .i,.;,.. =*.a.

_.:;:;r- ^>,< ’ “- , _:’ , ,, ^ . . .: -i IA description of Figure 3-l 1 1 3.46them are some centrifuge tubes (51), some test tubes(52), a funnel (53), a loop (54), a grease pencil (55), anda diamond pencil (56).Near the end of the bench is a microscope lamp (57)of the kind described in Section 6.11. This has a 12-voltbulb inside it and is joined by wires (58) to a car battery(59) underneath the bench. This battery is charged in theambulance belonging to the health centre. The wires arejoined to the battery by big clips (60). There is anOlympus Model K microscope under a plastic cover(61). Beside it is a booklet of lens paper (62) and a bottleof immersion oil (63). Right at the end of the bench aretwo of the locally made beakers described in Section3.11. One of them (the taller one) holds graduatedpipettes, the other (64) holds Pasteur pipettes.At the left of the bottom shelf is the Lovibond comparator(65), and in it are two Lovibond tubes. In one ofthese tubes there is a blood pipette (66) with a rubbertube and mouthpiece. Next to it is the haemoglobin disc(67) inits plastic box. The disc is kept in its box so as tokeep the glass standards clean and free from dust. Itshould be kept in the dark in a drawer in case its glassstandards fade in the light. On the back of the shelf is aglass jar containing glass chips in spirit (68). These arepieces of broken slides and are for taking blood from theear or finger (see Picture B, FIGURE 4-3). Next to it is.ajar full of pieces of cotton wool (69). These are used to stopbleeding from the hole made by the chip. There is also adropping bottle filled with spirit. This is for cleaning afinger or ear before it is pricked with a glass chip. Nearby is the counting chamber (70) with its cover glass ontop. Some people store their counting chambers in a dishof spirit. Beside it there is a box of ordinary thin coverslips(7 1). At the back of the shelf is the plastic tile (72)with depressions. In the health centre this is used fortesting the urine for INH. In a hospital it is also used forblood transfusion. There is a box of filter papers (73), alOO-ml stoppered measuring cylinder (74), a spirit lamp(75), and a jar of slides in spirit (76). The l,OOO-mlunstoppered plastic measuring cylinder is not shown.The slides in spirit are new ones and are mainly formaking blood films and films for AAFB. In front of thejar of slides in spirit there is a box of old and scratchedslides for making stool films (77). In the middle of theshelf is a spatula (78).The first two bottles of the row on the right of thebottom shelf (7) are ‘polystop’ bottles. One containsLugol’s iodine (79) and the other saline (80). Then comewash bottles with Benedict’s solution (8 1) and haemoglobindiluting fluid (82). In a hospital where bloodtransfusion is done a wash bottle of saline would also bevery useful. The dropping bottles contain 10% bariumchloride (83), white blood cell diluting fluid (54),Ehrlich’s reagent (85), Fouchet’s reagent (86), 20% sulphosalicylicacid (87), Pandy’s reagent (88), ferric chloride(89), saturated sodium acetate (90), and xylol (91).At the right-hand end of the bottom shelf are a bottle ofRothera’s reagent (92) and two Westergren ESR tubeshanging up on cup hooks (93). Some laboratories dotheir ESR’s by hanging them on hooks. You are providedwith a stand (ML 11). Next to them is a copy of thisbook (94). Hanging on the right of the front of thebottom shelf are a little tin of partin wax and Vaselineand a piece of bent wire. These are for sealing the edgesof coverslips.On the middle shelf are l,OOO-ml polythene reagentbottles of Benedict’s reagent (95), strong carbol fuchsin(98), Ehrlich’s reagent (99), form01 saline (loo), haemoglobindiluting fluid (lOl), Pandy’s reagent (102), andsaline (103). Next come several small things. There is asmall paint brush (105), a bottle of ink for the spirit pen(106), a tin of quick-drying paint (107), the spirit penitself (108), a pair of pliers ( 109), a hammer ( 1 lo), and apair of scissors (111). The best way to keep tools is tohang them from hooks on a board on the wall. Paint theshape or shadow of the tool behind the place where ithangs. If someone takes the tool away the shadow willtell you immediately that it has gone.At the right hand end of the middle shelf is a metal pan(112), a paraffin pressure stove (113), and the prickersfor it (114). Next comes the Ohaus balance (115) withone of the plastic watch glasses on its pan ( 116). Near itis the scoop (117) that goes on the pan and the extraweights ( 118).On the top shelf in alphabetical order are the bottles ofchemicals (119) that you will find listed in Sections 2.4and 13.10. There are boxes of test tubes and specimenbottles ( 120) and several old record books from last yearand the years before (12 1). Smaller bottles of liquids,such as immersion oil, can be kept on a high shelf. Biggerbottles of liquids such as methyl alcohol (122), spirit(123), hydrochloric or sulphuric acid (124), and xylol( 125) are best kept near the floor. They are less likely toget knocked over if they are on the floor.In this chapter we have tried to describe most of thethings that you will need. You will, however, find otherthings useful, so try to get them. These include small jars,par&n, matches, urine specimen jars, toilet paper, newspaper,etc.QUESTIONS1. Describe the ways in which you can supply waterto your laboratory bench.2. What is a filter pump and how would you use it?3. How does a Bunsen burner work, and what kinds offlame does it give?4. How is it possible to make use of the electricityfrom a car in a laboratory?5. &v; :~~ld you make a Pasteur pipette on a par&fin pressure :$tove?6. How d’u you make a wire loop? In what ways is agood loop di fferent from a bad loop?7. How do you (a) hold a hot test tube; (b) fold a filterpaper; (c) label a bottle neatly?8. What kinds of phosphate do you know? What arethey used for?

,,,I ,I. ,y,+;,,: c- ‘-> _ ,~“, ”3 1 Making the Laboratory Ready9. What are the following chemicals, reagents or teststrips used for: (a) Pandy’s reagent; (b) Lugol’s iodine;(c) Methylene blue in acid alcohol; (d) 20% sulphosalicy-lit acid; (e) 3.8% sodium citrate solution; (f) saturatedsodium acetate; (g) PAS test strips; (h) Congo red testpaper; (i) ortho-tolidine;(j) sodium carbonate; (k) for-malin; (0 para-dimethyl-amino-benzaldehyde; (m)phenol; (n) copper sulphate; (0) ‘Teepol’?10. What is distilled water? How is it made and whatkind of water can be used if it is not available?

4 1 Records and Specimens4.1 Records and reportsWhen you have found something out about a patient, say,for example, that he has hookworm ova in his stool, thisknowledge must be written down or it will be forgotten.You must write it down and so keep a record in thelaboratory of the hookworm ova that you have found.This record has to be kept in a record book, or recordfile. Used like this the word file means a special way ofkeeping papers, and is either a box, a drawer, or a book.As well as making a record in the laboratory, you mustalso report what you have found to the person who islooking tier the patient, so that he can be treated. Reportsare usually sent out of the laboratory on smallpieces of paper called report slips. These report slipshave then to be stuck or stapled to the patient’s notes (astaple is a small wire clip). This is better than copying thereport slips on to the patient’s notes, but, whatever isdone it should be done the same day. Only by doing thiscan we be sure that a labratory report will always be ina patient’s notes, and will not be lost.Specimens are often sent to the laboratory withanother small piece of paper called a request slip, whichtells you what methods to do on the specimen. Sometimesthe same kind of slip is used for both the requestand the report, as in Picture A, FIGURE 4- 1.4.2 Records for health centres and outpatientdepartmentsPatients who are treated in a health centre or a hospitaloutpatient department usually have medical notes thatare written on a sheet of paper or card. We will call thisthe outpatient or health centre card. This is often sosmall and thin that there is no place on it to stick a reportslip. The laboratory report has therefore to be copiedon to it in ink or stamped on to it with a rubberstamp.In small laboratories, when there are few specimensto examine, it may be convenient if patients leavetheir outpatient or health centre cards with their specimensin the laboratory. The laboratory report can thenbe stamped on these cards and in the report book.Patients can call later for their cards and find the reportalready written on them. This is what is being done at thehealth centre shown in FIGURE 3- 11.In larger laboratories, where many specimens areexamined from outpatients, it may be best if the outpatientleaves his specimen in the laboratory with a requestslip. The report is stamped in the report book, andthe request slip is thrown away. Next day, when thepatient comes back he hands his outpatient or healthcentre card into the laboratory. While the patient waits, aclerk looks up the report in the report book and copiesor stamps it on to the outpatient or health centre card. Itis very important for the laboratory report to be writtenor stamped on the health centre or outpatient card by thelaboratory stafl If the report slips described below areused, they soon become separated from the outpatient orhealth centre cards and are lost.The laboratory in a health centre or outpatient departmentcan use the rubber stamps that are described below.These rubber stamps can be stamped on the patient’snotes and in the report book. But, because no requestslips are filed, the report book (Picture 3 1, FIGURE 3- 11)must be ruled with enough space in it to stamp in thereport on each specimen.4.3 Records for hospitals (FIGURE 4- 1)The medical notes of patients in a hospital ward(inpatients) are usually written on several larger sheets ofpaper and are kept in a file like that in Picture D (here afile means a thin book). Because the medical notes ofinpatients are much larger than those of outpatients thelaboratory reports of inpatients can be kept differentlyfrom those of outpatients.What usually happens is this. When a doctor wants atest done on one of his patients in the wards, he writes itdown on a request slip. This request slip is then sent tothe laboratory with the specimen. The specimen isexamined in the laboratory, and a report is sent back tothe wards written on a report slip.Some hospitals use a different slip for the request andthe report, but it is easier to use the same one. Reportslips must be small, so that they take up little space in thepatient’s file of medical notes. A combined slip of thiskind for the request and the report is shown in Picture A.

4’ 1 Records and Specimens\ MARYA THIS IS THE REQUEST AND REPORT SLIP+--- -_ L5CE - - - ___ --*rI5cmI Haemoglobin ...._. ........._._._....._............ g1 BLOOD9this is a date stamp, it isstamped here on the sliptheseareyourinitialsthis is thelaboratorynumberTHESE ARE SOME OF THE RUBBER STAMPS THATYOU SHOULD HAVE MADE FOR YOUR LABORATORYEHaemoglobin.. ........................ g %Sickle cell test ..............................Total white count.. ........... cu mmPolymorphs.. ............................. %Lymphocytes ........................... %Monocytes.. .............................. %Eosinophils.. ............................. %, ............. ........................................I1 ................... .. ... ....._. .... ..................11 .......... ............,.......... .................. IBLOODF[-ElProtein .._.___._........................ mg % iCell count ......................... cu mmGram film.. ...................................II1 .. ............. ...... ........... ...... .. .... 1stamp.twe the rubber stamps made oninrooden blocks, they are cheaperwhen made like thisTHESEARETHEPATIENTNOTESthis is a file ’ - only this writing can be seenunless you lift up the slipsI Taenia ........................... ..............1 ;-;C;F;CM-- (~~~~~hod~~~~~~.~~~,......................... 1AAFB were seen in the following placesLesions (1). ........ Buttocks or thighs.. ..............,a(2). .....................................................II(3). .....................................................Ears, R.. .. L.. .. Bacteriological index.. ..........Nose.. ............ Morphological index .............saline srrear/thlckIURINEsmear/form&etherFig. 4-1Rubber stamps for recordsIt is a piece of paper about 15 cm long and 5 cm wide.Printers will often give away free the small pieces ofcoloured paper (offcuts) from which you can make theseslips. If possible use a different colour for each kind ofspecimen, say red slips for blood, blue for CSF, etc. Onthe top of the slip there are boxes for the patient’s name,his ward, and his hospital number. Underneath there isan empty place for stamping or writing the report. If youcannot get these boxes printed on the slips, have a rubberstamp made and stamp them on with this. Rubber stampssave much writing, and some of the stamps you shouldhave are shown in FIGURE 4- 1. Only the figure for theanswer is written. All other words are on the stamp.The date can be stamped on the slips with a datestamp. The best way to put the laboratory number on theslips is to use a paging numerator (page numberer). Thisis a special rubber stamp which can be adjusted to stampa number, one, two, three, or more times, before it moveson by itself to stamp the next number. Laboratory numbershelp to stop specimens being muddled and areespecially useful if the laboratory is a large one.The ward staff should stick report slips into thepatient’s notes one on top of the other, so that only theirbottom edges can be seen. You will see that the kind ofspecimen (BLOOD, CSF, etc.) is on the bottom of eachstamp, so that it sticks out below the bottom edge of thenext slip. The date and the laboratory number are alsostamped at the bottom. In this way the slip for a particularspecimen can be found very easily.If your laboratory is big enough to have several peopleworking in it, a good way to keep your records is theway shown in FIGURE 4-2. We will take a request for thehaemoglobin as an example. Specimens come into thelaboratory with their request slips through a hatch(Picture A-a hatch is a window for putting thingsthrough). The first thing to do is to make sure that the

; -. . . _specimen is lahelled and that the name on the specimen isthe same as the name on the form. The name and theward of the patient are written in a record book and on areport siip (Picture B). The kind of specimen is aisowritten in the report book. The laboratory number isthen stamped on with a paging numerator (not shown inthe figure). This is set (adjusted) to stamp the same numberthree times (once on the request slip, o&e on thereport slip, and once in the record book). The pagingnumerator will then move itself on one number and beready for the next specimen. If you have no pagingnumerator, the laboratory number can easily be writtenin with ink, but this takes more time.The request slip and the report slip are then stampedkith the right rubber st:bmp for that particular kind ofspecimen, dated with the date stamp and clipped together(Picture C). The date is only put into the record bookonce at the beginning of each day’s work. The specimenand two forms then go to the bench where the test isRecords for hospitals 1 4.3being done (Picture D). The haemoglobin of the bloodspecimen is measured (Picture E), and the answer iswritten in on both slips (Picture F). Both slips then goback to the record bench and are separated. The reportslip (Picture H) is placed on a nail in a special rack wherea nurse from the ward can fetch it. The request slip(Picture G) is filed alphabetically in a special box madeto fit it. Pieces of card are put hetween the slips of namesof patients which start with a different letter.There are two boxes for request slips, one for specimensexamined ‘this month’ (Picture I) and one for specimensexamined ‘last month’ (Picture J). On the first dayof every month the forms in the older of the two boxes ofslips are tied up with string and put away in a parcel(Picture K). If they are wanted (which is seldom) theycan always be found. The empty box is then used tofile slips for the coming month.Records of this kind are simple and cheap. There islittle writing to do, recent reports are easily found, and athe slips are then separated, the reportslip goes on the nail in the hatch/these are shelves with a placefor the renort slios for eachward : each place has a nail tostop the slips blowing awaya specimen of blood 0and a request sliphave just been broughtto the laboytory-thisis a hatch into the laboratorythis is the book withpatient’s ‘bloodgroups in ithis is a shelf. tc put the specimens o.n:the patient’s name, ward and numbecopied onto the second slip, the lanumber is written on both of themare then stamped with the ‘Haemoof the way somewherethese are the bundles of forms from previmonths kept in case someone wants to loat them againboth slips and the specimen go to theTHE HAEMOGLOBiNIS MEASUREDFig. 4-2Records for hospitalsBlood specimen with‘Mary Banda’ written

::, .: ,: . .4 1 Records and Specimensreport slip can be sent to the ward as soon as the answeris known. The only reports that should be copied into abook are the reports of blood groups. These are sometimesasked for months or even years later.Lf the laboratory has a telephone, the record bench isthe place to put it.Never send a report out of the laboratory withoutkeeping a record of it in the laboratory.4.4. The plus notationIn some of the methods in this book we measure somethingand give the report in numbers. For example, wemight report a patient’s haemoglobin as being 10 g %.But very often we do not measure anything. For example,we do not measure the protein in a patient’s urine.But in some specimens there is much protein and inothers only a little protein. We need some way of reportingthis, so we use what is called the plus notation.Notation means ‘way of writing’. Instead of using wordslike little, much, scanty, a few, or very many, etc., thereport is given like this:- = Negative ++ = Moderate+ = Doubtful +++ = Severe+ = Mild ++++ = GrossThe meaning of the words ‘positive’ and ‘negative’ isdescribed in Section 1.3. Report - or negative if you findnone of the thing you are testing for or looking for. Somepeople do not like putting a dash or ‘-’ sign for negative,and prefer to write ‘neg.‘, or to put a ring round the dashsign instead. Report + or doubtful whenever you are notsure if you have found what you are looking for. If youget a doubtful answer it is often wise to do the methodagain. When you are sure you have found what you arelookingfor, but there is very little of it, report +. Whenthere is a very large quantity indeed of what you arelooking for, report ++ + +. Use + + and + ++ whenthere are amounts in between + and +-t--c +. These haveto be judged, and different people will not always reportthe same result in the same way. Even so, this is a usefulway of reporting many of the methods in this book. Hereare some examples:AAFB ++++ means that there are very very largenumbers of acid fast bacilli in the film (see Section 11.1).Malaria parasites + means that you are quite sure thatthere are malaria parasites in the film, but there are veryfew.Bilirubinn + means that you are not sure what thecolour you see on the filter paper with Fouchet’s methodmeans. You have done the test again and are still doubtfuland don’t know whether to report the test positive ornegative (see Section 8.8).Form01 gel test - or neg., means that the serumremained clear and did not go solid when you addedformalin (see Section 7.40).Pandy’s test ++ means that the CSF became cloudywhen you added Pandy’s reagent. The mixture is morethan just cloudy +. so you report it ++ (see Section9.10).Trypanosomes +++ means that there are many trypanosomeson the film, but that you have seen filmswhere there were even more of them (see Section 7.36).The plus notation is a useful way of reporting things.But always give your report as a number lryou can. Forexample, it is better to make a standard faecal smear andcount the number of hookworm ova than it is to report‘Hookworm ova +‘.4.5 Preventing mistakesEarlier in this book you read about how important it is totell the truth. It is also very important to try to preventmistakes. There are many reasons for mistakes, but hereare some of the things you can do. Many of them areabout records.METHODPREVENTINGMISTAKESAlways label any slide or tube with the patient’s namebefore You start a method. Never accept an unlabell@dspecimen from the ward or bring an unlabelled spacimeninto the laboratory yourself. When a specimen isbrought to the laboratory, check that the name on therequest slip is the same as the name on the specimen.Check this before the person who brought the specimenleaves the laboratory. If they are wrong, hand themback. Never accept a specimen without a request slip.Be very careful not to mistake names, especially commonnames like Pate1 in parts of India, Musoke inBuganda and Banda or Phiri in Zambia. Try to use twoinitials or two names for patients with common names,such as K. Y. Patel. A. B. Musoke, Y. N. Banda, L. S. Phiri.One of the main uses of a hospital number is to preventmistakes from muddled names. Whenever you can,try to use the hospital number, or at least the last threefigures of it.All these rules are especially important with specimensfor blood transfusion. Blood of the wrong groupmay kill the patient.4.6 Specimen containersSometimes patients come to the laboratory themselves.and you can take specimens from them yourself. Moreoften specimens are taken from the patients by nurses ii1the wxrds and are sent to the laboratory in containers(bottlt!L; or boxes). A laboratory must therefc3re get thy:empQ containers ready and send them to thl: wards.Several kinds of containers can be used for sprzimen?,.Some are she\\ n in’ FIGURE 2- 1 as ML 14 a, b, c, and ci.ML 14a is a small glass bottle called a bijou bottle. It hasa metal cap and a rubber washer or liner. 14b is a largerglass bottle called a universal ccretainer; it has the samekind of cap and liner. ML 14c is a plastic containercalled a polypot. ML 14d is anotl‘zr plastic containercalled ;L polytube. Bijou bottles cost $0.06, and universalcontainers cost 150.08: so they are both quite expensive.But Folg’pots only cost $0.023, and polytubes only cost

“‘MAKING A SEQUESTRENEPOLYTUBEAMAKINGGLASS CHIPS&G3cap ofPolVtubea little bit ofseques’trene inthe bottom ofT’.C PolytubeWRAPPING UP A STERILESYRINGE AND NEEDLE:F“eed,ebarrel ---y&-y&qp$ .vnozzle y,this slide has beenL--I.-- --*- -L.--orcmen 1nro cmpschipsthe ch=ept in abottle of 70% spiritthis is a heat sealingplungermachinethere is a trssue soecimen 2. the parts of the inside this bagsyringe are separatevnu will find ileasier to use alget the chips outmade : the cottonwool is flattenedout, the sides arefolded into themiddle and it isthis is a large /sharp needleSEALING A TISSUE SPECIMEN INSIDE A POLYTHENE BAGHthese parts of theare heated by elec._U.-?.- 3.\wirec?this is the syringe already ,wrapped up for~autoclaving \in the pressure cookerGthis is a roll ofpolythenetube : itis verv thin and isrolled up flat/-the jaws of the machineclose and seal up thebagthis is the pedalfor the machineTAKING BLOOD FROM A FINGERTAKING BLOOD FROM AN EARyour left handthe patient’sfingerprick into the edge of the ear-ALlass chip3wFig. 4-3 Specimen containers

4 1 Racords and Specimens$0.0075; so they are both quite cheap. Because all kindsof container so often disappear in the wards, keep expensiveglass bottles in the laboratory. Send cheap plasticcontainers to the wards, so that if they are lost they willcost less to replace. Polypots can be boiled and autoclavedbecause they are made of a special kind of plasticcalled polypropylene-see Section 1.3. The polxytubeslisted in this edition can only be boiled.Polypots are used for stools, urine, and sputum. Threekinds of polytubes will be needed for blood.Plain emp@ polylubes. These are used for clottedblood. This is mostly used for blood grouping and crossmatching. These are not very common methods, so fewempty polytubes will be needed.Sequestrene polytubes. These are for anticoagulated(unclotted) blood, and many will be wanted. They shouldhave a very small quantity of sequestrene in them, asshown in Picture A, FIGURE 4-3. Sequestrene is an anticoagulantwhich will stop blood clotting-see Section1.17. If you have no sequestrene you may be able to useWintrobe’s mixture instead. Weigh 12 g of ammoniumoxalate, mix it very well with 8 g of potassium oxalate.Make sure the mixture is finely powdered and use it justas you would sequestrene.Potassium fluoride polytubes. Potassium fluoride stopsthe cells of the blood or CSF using up sugar. These polytubesare therefore only used for measuring the sugarin specimens of blood or CSF. As with sequestrene, onlya very small quantity of potassium fluoride is required.The easiest way to tell one kind of polytube fromanother is to mark them with a spot of paint. It does notmatter what colour is used, but it is helpful if all hospitalsin a country use the same colour. If you can choose,leave plain polytubes unpainted. Put a spot of yellowpaint on the sequestrene polytubes, and put a spot of redpaint on the fluoride polytubes. There are tins of red andyellow paint for this in the main list of equipment (ML64). Spirit pens can also be used.Polytubes, polypots, and slides can be labelled with agrease pencil, but the wards may not have grease pencils,so you should stick a paper label on to each tube withpaste or gum. Polytubes can be bought already labelledand filled with potassium fluoride or sequestrene (MBO).but they are more expensive than making your own.The nurses in the wards will want to know whichspecimen to put into which bottle; so put a notice in eachward to tell them. It will also help if you put notices inthe wards about any other things you think the ward staffshould know, such as when to send specimens or how tomake thick blood films.Here are some instructions for you to copy out andput on the ward notice boards. They are given you as amethod.METHODA NOTICE FOR THE WARD NOTICE BOARDSPlain PO&tubes (no paint spot). Use these for theblood grouping, cross matching, and the formal gel test.Red spot PO&tubes (fluoride). Use these for the bloodsugar and CSF sugar.Yellow spot Polytubes (sequestrenel. Use these forthe haemoglobin, the white cell count (total and differential),and the sickle-cell test, the ESR. concentrationtests for microfilariae and trypanosomes (send the bloodto the laboratory immediately), the blood urea, and thereticulocyte count.Thick films. Make your films like this (stick a goodthick film to the notice board, as described in Section7.31 and cover them with polythene sheet to keep awaythe flies).Please send your specimens to the laboratory early inthe day.4.7 Capillary and venous bloodFirst you must learn a little about how blood goes roundthe body. Blood carries food and oxygen (from the air) tothe tissues of the body. Without this they cannot growand work. Blood is pumped (pushed’or sent) by the heartalong tubes with thick walls called arteries. When we feela patient’s pulse, we can feel the blood being pumpedalong an artery by the heart. The blood in an artery isunder pressure and will rush out (bleed) very fast if theartery is cut. Blood from an artery goes into many verythin-walled tubes called capillaries. These are so smallthat they can only be seen with a microscope. There arevery many capillaries in every tissue of the body includingthe skin. While blood is in the capillaries it givesoxygen and food to the tissues and takes waste substancesfrom them. If any part of the body is cut, itbleeds because these capillaries have been opened.Capillaries join together to form larger thin-walledtubes called veins. You can easily see the veins of theskin, especially those in the arm and on a warm day.Blood goes along the veins back to the heart. Blood is notunder much pressure in the veins, and they easily go flatand empty. Blood coming in the veins from the tissues ofthe body is pumped by the heart through arteries to thecapillaries of the lungs. Here it takes up oxygen from theair and gives up a waste gas called carbon dioxide. Bloodcomes back from the lungs to the heart In veins. Theheart then sends this blood full of oxygen through thearteries to all parts of the body.Often, by testing a patient’s blood, we can find outmore about his illness. It is difficult to get blood fromarteries, but it is easy to get blood from veins and capillaries.Blood can easily be taken from one of the veins ina patient’s arm. A rubber tube is tied around his arm topress on the vein and shut it. Look at FIGURE 12-7. Thistube stops blood in the vein getting back to the heart andmakes it fill up with blood and swell (get bigger). Aswollen (big) vein like this can easily be seen, and youcan easily feel it with your finger. Blood can be taken outof a swollen vein with a syringe and needle or with arubber tube and needle. Blood from a vein is calledvenous blood, and many millilitres can be taken. Venous:

Cross infection and syringe jaundice 1 4.8blood is also taken for blood transfusion and is describedin Section 12.12.Some patients, especially children, do not have veinswhich are easy to see and big enough to put a needle into.It is often useful therefore to take capillary blood instead.Capillary blood can be taken from any part of the body,but the easiest place to take capillary blood from is theball (end) of a finger or the lobe of an ear (see FIGURE7-l). The ear is less painful than the finger, and thepatient cannot see what you are doing and is thus lessfrightened. The heels of very young babies are also used.Special small knives called lancets can be used, so canglass chips (a chip is a small piece, see below). It isusually only possible to get a few large drops or about atenth of a millilitre (0.1 ml) of capillary blood. But this isenough for many methods.Glass chips are cheap, easy to use and safe. They donot make quite such a clean cut as the special sterilelancets that can be used, but they are safer and drawmore blood than the prick (puncture, stab) of a needle. Ifyou have these special small lancets, they can be sterilizedand used again.METHODGLASS CHIPS FOR CAPILLARY SPECIMENS, PICTURES B ANDC. FIGURE 43MAKING GLASS CHIPSBreak slides in the middle so that there are as manylong-pointed chips as possible. If you do not get goodshapedchips, try hitting the middle of the slide with ahammer. Make good use of all old broken slides in thisway. Collect the pointed chips and put them in a smallwide-mouthed screw-capped jar of 70% spirit (spirit70 ml. water 30 ml). This jar is shown as number SB inFigure 3-l 1. Use a small jar so that you can easily getthe chips out.TAKING CAPILLARY SPECIMENSTake a piece of cotton wool and put spirit on it. Keepa dropping bottle of spirit for doing this. Clean thepatient’s ear, or his finger, or the ball of his heel if he is ababy.Squeeze, or flick the ear, finger, or heel a few timesbefore you prick them. This will make more blood cometo the capillaries. Make sure that the skin is dry beforeyou prick it, and if necessary dry it with clean cottonwool. Make one short sharp prick as shown in Pictures Jand K in Figure 4-3. Blood will start to come and willsoon make a large drop. Try not to squeeze the finger orear after you have pricked it, because this spoils thespecimen for some methods.Make a blood film straight from the drop of blood orfill a blood pipette with it.Throw away the chip.It is sometimes easier to put a ver:): l’e,T Me sequestrenein the bottom of a small test tube (a ‘cross-matchingtube’, ML 48b) and catch the blood in this as it drops outof the prick on the ear or finger. Let each drop of bloodflow down the same side of the tube. They will reach thebottom of the tube more quickly and dry less readily. Ablood pipette can then be filled more easily from thedrops of blood at the bottom of this tube.4.8 Cross infection and syringe jaundiceThere is one very important thing to remember whentaking blood specimens from patients. It is this. Neverput a needle or glass chip into one patient and then intoanother patient unless you have sterilized it first. As youhave read, sterilizing something means killing all themicro-organisms on it, especially those which causedisease. If a needle is taken out of one patient and putinto another without being sterilized, micro-organisms inthe blood of the first patient may go on the needle intothe second patient and cause disease. Washing a needledoes not remove the micro-organisms. Only a very smallamount of blood need be left on the needle to make itdangerous-so little blood that you cannot see it. Manyharmful micro-organisms can be taken from one patientLO another with a dirty needle in this way. Malaria parasitesare one, but the worst micro-organism of all is avirus which causes a disease called syringe jaundice(serum hepatitis). Hepatitis is a disease of the liver. Jaundiceis a disease in which the patient’s body goes yellow.Patients often die from this disease. To prevent patientsbeing infected in this way NEVER PUT A NEEDLEINTO A PATIENT UNLESS IT HAS BEENSTERILIZED FIRST. NEVER, NEVER, NEVERPUT THE SAME UNSTERILIZED NEEDLE INTOMANY PATIENTS. Some people use the same syringefor each patient but use different sterile needles. This toois dangerous because blood from one patient left in asyringe can go up an otherwise sterile needle and intoanother patient. When one patient infects another in ahospital or health centre, the second patient is said tohave been cross infected. Cross infection must be prevented.Dirty syringes and needles are one importantway in which it can happen.4.8 Syringes and ‘needles and tubes’The best way to sterilize syringes and needles that aregoing to be used for taking blood is to autoclave them.For this a pressure cooker is very useful. Autoclavingnot only kills the micro-organisms but leaves the syringedry. Dry syringes are important because the water in awet syringe lyses some red cells and makes the spec;menuseless for many methods. But syringes that have beensterilized by boiling can easily be used for giving injections.because it does not matter if a patient is injectedwith a little sterile boiled water as well as his drug.Syringes for taking blood must not be kept in spirit,because spirit in the syringe is even more likely to causehaemolysis than water. Spirit can, however, be used forstoring glass chips. 70% spirit is an antiseptic and will

4 1 Records and Specimenskill the micrcForganisms on the chips. Spirit soon driesfrom the chip and does not get into the blood.Most glass syringes and some plastic syringes can beautoclaved in large test tubes, in special metal tins, orwrapped in paper. The next method tells you how tosterilize syringes wrapped in paper. Syringes made onlyof glass (‘all glass syringes’) are the best, but they areexpensive. If you have some, look after them carefully.METHODSTERILIZING SYRINGES. PICTURE F. FIGURE 4-3Make sure that the needle is sharp and not blocked(Pictures 0, P. and Q, Figure 12-6) and that the syringeis clean and dry. Dip (put) the end of the phnger (theplunger is the inside part of the syringe, the barrel is theouter part) into a little liquid paraffin. Liquid paraffin is athick clear medical oil. This oil will lubricate the plungerin the barrel (make it move easily).Cut sheets of thick brown paper into pieces of theright size to wrap a syringe. Put the needle into onecorner of the paper; fold this comer towards the middleof the paper. lake the plunger out of the barrel and wrapit up separately as shown in Picture F. If the plunger issterilized while inside the barrel, the barrel may crack.Tie the parcel up with string and steriiize it in thepressure cooker by the method in Section 1.21.When sterilizing is finished. write ‘sterile’ on the parcel.Also write the date.Syringes are expensive and are often broken, lost, orstolen. There are thus too few of them in many hospitals.Fortunately they are not always necessary, and it is oftenpossible to use a ‘needle and tube’ like that shown inPicture E, FIGURE 4-3. A large needle is fixed to a shortpiece of rubber tube. The needle and tube are then putinside a test tube. The test tube is plugged (closed orcorked) with a plug of cotton wool and sterilized in apressure cooker. Cotton wool plugs let air through, butthey keep micro-organisms out. After the tube has beensterilized no micro-organisms will get in unless the plugis removed.METHODMAKING NEEDLES AND TUBES, PICTURES D AND E. FIGURE4-3Take a large needI- 6-cm No. 21 gauge needle is agood size, but any iarge sharp needle can be used. Fixabout two inches of rubber tube on to it. Plastic tube willprobably melt in the pressure cooker. Put a piece of bentwire into the end of the needle and put it pointdownwards in a test tube. New needles are usually suppliedwith pieces of wire in them. The end of the wirewill stop the point of the needle getting blunt on thebottom of the tube. Cotton wool in the bottom of thetube can also be used.Take a piece of cotton wool. The size of the piece isimportant, and only practice will show you how muchcotton wool you need to make a good tight plug. Open itout flat. Fold the sides to the middle, as in Picture D.Then roll it up. This will make a tight. cork or plug for thetube. Autoclave the plugged test tube with the needleinside it in a pressure cooker. as described in Section1.21.To use this kind of needle, take out the plug andgently shake the needle part of the way out of the tube.Take hold of the needle where it is covered by the rubbertube between your finger and thumb. Don’t touch thepoint of the needle. Put the end of the rubber tube intothe bottle or tube that is to hold the blood. Put theneedle into the patient’s vein as shown in Figure 12-7.These needles and tubes are not so easy to use assyringes, but they are much cheaper. Keep some of themready to use. Wash them as soon as they have been used,and make sure that the points of the needles are sharp.As soon as they get blunt, sharpen them by the methodshown in FIGURE 12-6.4.10 Sending specimens to a central laboratorySome specimens can he sent from a health centre ordistrict hospital to a central laboratory. If specimens arenot to be spoilt before they arrive, it is very importantthat they are packed and sent properly. This section tellsyou how specimens should be packed and sent. Some ofthese instructions are for doctors.Histology (the study of tissues)Histology specimens are pieces of a patient’s tissueswhich are to be cut into sections (very thin slices) andlooked at with a microscope. A doctor takes these specimensfrom his patients in an operating theatre, but youmay have to wrap them up and send them to the centrallaboratory. Histology is usually only done in big centrallaboratories.As with all specimens, see that the patient’s name, age,sex, village, and tribe are put on the request form, as wellas something about his illness.All tissue for histology has to be fixed. That is, thecells from which it is made must be killed. The tissuemust also be prevented from putrefying (see Section1.15), and the cells from which it is made must be keptlooking just as they did when they were alive. Tissues arefixed by putting them in a fixative solution. Form01 salineis the illtibt btiiifiiiui::j Gsed fixative for tissues (seeSection 3.29). Other methods of fixation used in thisbook are methyl alcohol. as used in Leishman’s stain,and heat. Heat is used to fix the cells and microorganismsin films of sputum and pus.The most important thing to remember when fixingtissues in formol saline is to use enough formal saline.Use at least jive times as much form01 saline as there istissue. Form01 saline must also be able to get into themiddle of the tissue. If a piece of tissue is small formolsaline can easily get to all the cells. But, if a piece of

: 1 _;;:.,.“ -. y ‘, ;Send&g specimens to a central laboratory 1 4.10tissue is large, it must either be cut in slices, or specialparts of it must be cut off for sectioning. These specialparts are called blocks. Knowing where to cut theseblocks on a large piece of tissue, or how to slice it up, is ajob for a doctor. Only .he will know where to cut theblocks. He should cut Several blocks 3 x 2 x 1 cm fromdifferent parts of the tissue. If the whole of the tissue is tobe sent, it must be cut into slices, but they should be leftjoined together at one edge. Put the slices together againand leave them for several days in a bucket of form01saline, if possible in the cool of a refrigerator. Severallarge specimens can easily share the same bucket ofform01 saline while they are fixing. Tie a string to each ofthem, and put labels on the ends of the strings hangingoutside the bucket,Send small specimens in small, watertight (not leaking),screw-capped bottles. Universal containers can besent through the post in a strong envelope. Specimenscan also be sent in a polypot. Fix them first and packthem in the polypot with some cotton wool made a littlewet with form01 saline. Polypots may leak; so thereshould be no liquid inside them. Don’t send large screwcappedjars, because they often leak and break in thepost. If big specimens have to be sent, cut them in slicesand fix them well as described above. Then wrap themseveral times round in a polythene bag. Seal this bag withadhesive tape (sticking plaster). Don’t use staples becausethese make holes in the bag. Pack the tissue wrapped inits polythene bag in a cardboard box. You can also sendsmall specimens in a polythene bag like this, but put apiece of cotton wool soaked in form01 saline in withthem. This will stop them getting dry.The best way to pack and send tissues to a centrallaboratory is to seal (close) them inside a polythene bagwith a heat-sealing machine like that shown in Picture H,FIGURE 4-3. This sealer has two jaws (parts of a mouth)heated with electricity. When the hot jaws are closed,they make the polythene soft so that it sticks togetherand seals the bag. You will need the thin, flat polythenetube in a roll that is shown in Picture I. Cut off a shortlength of tube. Seal one end of this with the sealer. Thiswill make a bag. Put the tissue into the bag with someformalin, and then seal the other end of the bag.Some central laboratories supply district hospitalswith labelled wooden blocks. Each block holds a universalcontainer filled with form01 saline. This universalcontainer holds the specimen. These blocks have squarelids which pivot on a nail. The lids are fixed with ‘Sellotape’(plastic sticking tape) and the blocks are sent offunwrapped to the central laboratory. They are a verygood way of sending specimens. Put a pencil and papernote of the patient’s name and the date with eachspecimen. Pencil writing will not wash off in the form01saline, but most kinds of ink will.Serology (the study ofsera)There are many methods for examining serum which canonly be done in a central laboratory. The best ,way tosend a serum specimen is to take blood aseptically (seeSection 1.22) into empty sterile bottles and pack thesebottles with small pieces of ice in a vacuum flask. Avacuum flask is a bottle with two walls. The air betweenthese two walls has been taken away leaving nothing.Where there is nothing, not even air, we say there is avacuum. Heat cannot get across a vacuum. So if there issomething hot inside a vacuum flask, it stays hot for along time because the heat cannot get out. If there issomething cold, such as ice, inside a vacuum flask, itstays cold for a long time because the heat cannot get in.Because the inside of the vacuum flask stays cold forseveral days micro-organisms do not grow, and clottedblood specimens keep well.There is another way of keeping specimens cold. Thisis to use a box made of thick, light, white plastic calledexpanded polystyrene. The box is made cold with specialsold bags which are made cold in a refrigerator and areused instead of ice. Expanded polystyrene lets heat intothe box very slowly and the cold bags can be used manytimes. These boxes do not break so easily as vacuumflasks. Vacuum flasks are very easi!v broken. so lookafter them with great care.If you have no vacuum flask or &ld box, and theserum is going to take some days to iet to the centrallaboratory, it should be preserved (kept as it is) with anantiseptic (see Section 1.19). The best antiseptic for preservingserum is sodium azide. If possible separate theserum aseptically with a sterile Pasteur pipette and addto it a ver3pfeHt crystals of sodium azide. You can use asolution of sodium azide instead. Dissolve 0.5 g (500mg) of sodium azide in 25 ml of water. Add one drop oftbis solution to every ml of serum you want to preserve.Write on the form that you send with the specimen thatyou have added sodium azide.Bacteriology (the study of bacteria)The specimen that you will want to send most often issputum for the culture of Mycobacterium tuberculosis. Acentral laboratory may be able to find mycobacteria byculturing them (growing them in a special way) when youhave failed to find them by the Ziehl-Neelsen method(set Section I 1. I). A central laboratory can also find outwhich drugs can be used to kill the mycobacteria that areinfecting a patient. This is called sensitivity testing.Send the sputuum in a universal container inside astrong envelope, or wooden block (see above).Haematology (the study of the blood))Haemoglobin can be measured in quite old specimens ofblood. But white blood cells are soon destroyed. If thereforeyou want to send blood away to be looked at, make athin blood film and fix it with methyl alcohol. This iseasy--pour a few drops of methyl alcohol over the filmas soon as it is dry. By the time the methyl alcohol has

dried up the film will be fixed. When you write out therequest form, say that the fihn has been fixed. If you haveno methyl alcohol, stain the film with Leishman’s stain,and send the stained film instead.Films of the bone marrow can be sent in the sameway. But, if you send thick films for blood parasites, sendthem unfixed.Biochemistry (the study of the chemistry of livingthings)The only biochemical methods for blood in this book arethe blood sugar and the blood urea. There are manyother biochemical methods. Most specimens for biochemistrydo not travel well in the post. You can,however, send serum for the measurement of the serumproteins and the serum calcium. These specimens travelwell.ParasitologyThe word parasitology is usually used to mean the studyof worms and protozoa. It does not usually mean thestudy of bacteria and viruses even though these are parasites.Put large parasites in form01 saline. Preserve stoolsin two or three times their volume of form01 saline.SEND CAREFULLY FILLED IN REQUESTFORMS WITH ALL THESE SPECIMENS.QUESTIONS1. Why are records and reports so important?2. Describe the ‘plus notation’.3. How can mistakes be prevented in the laboratory?4. What kinds of specimen container do you know?What is good and bad about each kind?5. What uses do you know in a laboratory for: (a)potassium fluoride; (b) liquid paraffin; (c) sodium azide;(d) a broken glass slide?6. Describe the ways in which tissue for histologicalexamination can be sent to a central laboratory.7. What is meant by a capillary blood specimen? How.would you take such a specimen? For what sort ofmethods is capillary blood especially useful?8. Why is it so very important not to put the sameneedle into many patients without sterilizing it properlybetween one patient and the next patient?9. What is meant by the words ‘cross infection’? Whatpart can the staff in a laboratory play in preventing it?10. How can venous blood be taken without using asyringe? How would you prepare the equipment fordoing this?

5 1 Weighing and MeasuringWEIGHT5.1 WeightWhen‘ we buy something in a market we want to knowhow much of it we are buying, how much fish perhaps, orhow much oil. We need some measure for the weight ofthe fish and for the volume of the oil. Only if we useweights and measures can we be sure that we are gettingthe right amount of fish, or the right amount of oil. In thelaboratory we also want to know how much of somethingwe are using. We use the gram for measuring weight andthe millilitre for measuring volume. Grams and milli-Iftres are part of the metric system. The metric system iseasy to use because everything is in tens, hundreds, orthousands. Thus there are a thousand milligrams in agram, and a thousand millilitres in a litre (‘mille’ means athousand).The most important weight is the gram, which is oftenshortened and written ‘g’. Five grams, for example, iswritten 5 g. The gram is quite a small weight. A newpencil, for example, weighs about five grams. When youweigh out the chemicals for the methods described in thisbook, you will weigh perhaps 2, 10, or 20 grams. Thegram is thus a very useful siz;e of weight for a laboratorylike ours.But sometimes we want to weigh something smallerthan a gram, say half a gram or a fifth of a gram. At othertimes we want to use a weight that is in between wholenumbers of grams. For example, we might want to wei@something between three and four grams, say three and ahalf grams. We could use fractions, like ;t half(j) or aquarter (+) of a gram, but it is easier to use what arecalled decimals. The word decimal means tenths, and adecimal is only a fraction made into tenths. Thus a half ismade into five tenths, and a fifth is made into two tenths.To make it easier to write these decimals we leave outthe ten and write a dot called the decimal point instead.Two-tenths become -2 and five-tenths become .5. Forexample, forty-three and two-tenths becomes 43.2. Infront of the decimal point are the whole numbers ofgrams, such as 43 in this example. After the decimalpoint come the tenths of a gram, such as -2 and -5 inthese examples. When there are no whole grams ‘0’ isusually put in front of the decimal point. Half a gram isthus written O-5 g and two-tenths of a gram, or ‘pointtwo of a gram’ is written O-2 g.It is often useful to use a smaller weight than a gram.The weight that we use is the milligram. There are athousand milligrams in a gram. Milligrams are written‘mg’. Thus half a gram, or O-5 g is the same as 500milligrams or 500 mp. A fifth of a gram or 0.2 g is thesame as 200 mg. A tenth of a gram or O-1 g is the sameas 100 mg. The methods in this book sometimes require500 mg or 100 mg of a chemical, but we do not needsmaller quantities than this.5.2 The Ohaus triple beam balanceThe balance shown in FIGURE 5-l is called the Ohausbalance after the factory where it is made. As you willsee it has a large round pan for holding things, and threearms or beams-triple means in three parts. At the backis an extra beam, or fourth beam, called the tare beam. Itis used for balancing the watch glass, cup, or paper thatare used to hold a chemical while it is being weighed. Oneach beam there slides a weight and at the end of thebeams there is a pointer. The beams and the pan move ona hinged part of the balance called the pivots. When thebalance is adjusted the beams swing so that the pointercomes to rest opposite a line on the pillar of the balancemar&d ‘0’. Before anything is weighed the balance mustbe adjusted so that the weights on the front three beamsare set at ‘O’, and the pointer also points to ‘0’. This canbe done by turning the poising nut in and out until thepointer points to ‘0’.In this balance weighing is done by sliding threeweights along the three beams. The front weight is forweighing things up to IO g. The back weight IS forweighing things up to 100 g. The middle weight is forweighing things up to 500 g. These weights are oftenused tclgether. If you look at the front beam you will seethat the space between each whole gram (the space between1 and 2 g, for example) is divided into ten :’ : 1 I 3.The balance can thus weigh tenths of a gram or 1 S,‘: I‘ iIf you look at the front beam in Picture B you \!,iil v.(>that the front pointer points to 1.1 g (one gram aiL .I I.mg). Because it is not very easy to see this in Pictrarc B.the front weight has been drawn again much bigger in

5 1 Weighing and MeasuringAextra weightsthis weight is at 2LEFT8this is a view lookingdown from on topthese dotted lines show theplace of all the weightsi-:is poised thepointer shouldbe at ‘0’. itis a bit lowin this picturehereRIGHTi/I//pan\\\’\\\\-rromwelgnrextra weights’hang hereeach of theseis 100 mgdivisions.this is to show thefront weight weighingFig. 5-1The Ohaus balance

The Ohaus triple beam balance 1 ‘5.2Picture C. The back weight points to 90 g. The middleweight points to 300 g. If there was something in the panand the beams were poised, the balance in Picture Bwould therefore be weighing 300 + 90 + 1.1 = 39 1.1 g.In Picture A the front weight points to 2 g exactly.The back and middle weights are at 0. This balance willtherefore be weighing 2 g exactly.You will see that the front weight can slide anywhereup and down its beam. But the back beam has notches(pieces cut out of the beam) for each ten grams, and themiddle beam has notches for each hundred grams. Theback and middle weights must always be in these notches.If you want to weigh 150 g, put the middle weight in the100 g notch and the back weight in the 50 g notch. Don’tput the middle weight halfway between 100 and 200 g andthii that it will weigh 150 g. This will not be accurate.Chemicals must never be put straight on to the pan ofa balance, because this might spoil it. They have to beweighed on a watch glass, or on a piece of paper, or in acup. Because a watch glass, or a cup, or even a piece ofpaper weigh something, it is useful to have the tareweight to adjust the pointer to ‘0’ with.The balance is magnetically damped. By this we meanthat magnets are used to slow the swinging of the pointerand bring it to ‘0’ more quickly. These magnets are in thepillar and a piece of metal fixed to the beam swings inbetween them. It must not touch these magnets, and if itdoes touch them, gently bend it away from them.(c) USING THE TARE BEAMMake sure that all the weights are at ‘0’ and that thebalance is poised. Make sure the pan is clean.Put a watch glass on the pan (you may sometimesfind it useful to use a plastic cup instead of a watchglass). Move the tare weight along the tare beam untilthe pointer swings to ‘0’ and the kalance is again poised.You will now have ‘tared’ the watch glass.(4 WEIGHING A CHEMICALAs an example, let us say that you want to makeBenedict’s reagent as described in Section 3.18. Youwill want 17.3 g of copper sulphate.Poise the balance.Tare a watch glass as described above.Make sure the middle weight is in its ‘0’ notch. Movethe back weight to 10 g and see that it is also in itsnotch. Move the front weight to 7.3 g. This will be 17.3 gin all. The beam will go down while you do this.With your spatula put some copper sulphate in thewatch glass. Go on adding more and more until the panfalls and the beam rises. Then take some copper sulphateoff the watch glass little by little until the pointerswings around ‘0’. By adding more copper sulphate ortaking it away you will be able to get the pointer toswing on ‘0’. The beam will now be poised once more,and you will have weighed out exactly 17.3 g of coppersulphate.(4 USING EXTRA WEIGHTSMETHODUSING THE OHAUS BALANCE. FIGURE B-lM UNPACKING THE BALANCETake the balance out of its box. The weights arepacked in another part of the box. Make sure you findthem. Several pieces of rubber called the beam retainers(holders) are fixed to the balance to hold the beam while itis in the box. Take the beam retainers off the balance andkeep them. You may want to pack the balance again.Put the large weight on the middle beam. Put thesmall weight on the back beam.VII POISING THE BALANCEMake sure that the pan is empty and clean and thatthe balance is on a flat level surface.Move the poising nut to the middle of its screw.Push all the weights, including the tare weight, as faras they will go to the left. They will be in the placesshown by the dotted lines in Picture B. They should allbe at ‘0’. The back and middle weights must be in theirnotches.Make sure that the pointer swings easily and is nottouching the side of the stand. You may have to move itforward a little.Move the poising nut until the pointer swings to ‘0’ onthe scale. The balance will now be poised. ALWAYSMAKE SURE THE BALANCE IS POISED BEFORE YOUWEIGH SOMETHING.If you are weighing something which is more than610 g, you will have to use the extra weights which areshown in Picture D, Figure 5-1. These weights weigh1,000 g and 500 g on this balance, but their real weightis much less, because they are being used at the end of along beam. They will not weigh 1,000 g or 500 g on anyother balance. Hang them in the places shown by thedotted line in Picture A. Add the weights shown by thethree beams to the extra ones you add on. The heaviestthing you can weigh is 2,610 g. This will be using thetwo extra weights of 1,000 g each and all three weightsright at the end of their beams. You will not need to usethe extra weights for the methods in this book, but theyhave been put in the equipment list because they makethe balance more useful. You may want to use them forother things in a hospital or health clinic. A scoop hasalso been put on the list. It is really only like a very bigwatch glass and is for weighing things which are too bigto go in a watch glass or a plastic cup. Tare (balance) thescoop with the special counterweight for it, and use itjust as you would a watch glass.(f) LOOKING AFTER THE BALANCEWhen you are not using the balance, keep the panempw.Keep the balance clean.Never oil the balance.When you move the balance, move it carefuIly%n6make sure it is not hit or dropped.

5 1 Weighing and MeasuringA TWO EVERYDAY MEASURESTEASPOa spoonful ofwater is aboutTHIS IS USEFULROUGH WAY TOMEASURE 5 MLthese are allmade of glassGRADUATEDPIPElTESGfiML320.1 ml ’fill a test tube astmgers and it willhold about 5 ml3 mrtwofingers0.1 mlBLOODPIPETTEa plasticM ;z;;p;;z$;ry-s.-1000 mlMEASURINGCYLINDERSa glass lOOmI measuringcylinder with a stopper1000mlKiESE ARE THE BOTTOM PARTSF THE PIPETTES DRAWN LARGERFig. 5-2 Measuring the volumes of liquids

: ,!“&;‘.: ; ), : IIPipettes and measuring cylinders 1 5.7If your laboratory has shelves like those in FIGURE 3-11, keep the balance on the lowest of them, not on themiddle one, as shown in this figure. You will be able touse it while it is on the shelf, and it will not take up anyroom on the bench.VOLUME5.7 Pipettes and measuring cylinders (FIGURE 5-2)We measure volumes for two reasons. Sometimes wewant to find out how much space or volume is filled bysome liquid we already have. More often we want to takea special volume of some liquid, so that we only haveexactly as much liquid as we want. This is the reasonwhy we measure liquids in the methods in this book. Inthe first section of this chapter you read that the volumesof liquids are measured in millilitres or ml. Some peopleuse the word cc or cubic centimetre. A cc is almost thesame as an ml, but it is much better to use ml. A cupholds about 200 ml, and a teaspoon holds about 5 ml. Ifyou take an ordinary test tube and fill it as deep as twofingers are wide, it will also hold about 5 ml. These usefulbut inaccurate ways of measuring liquids are shown inPictures A, 13, and C.In a medical laboratory we measure liquids by puttingthem into special bottles or tubes which are made to holdexact volumes. These have graduation marks (see Section1.3) on their sides to show how much liquid is in themwhen the top of the liquid is at the graduation mark.Some of the measures we use have only one graduationmark on them, some have many graduation marks. Weuse measuring cylinders, graduated pipettes, and graduatedcentrifuge tubes.There are two measuring cylinders in the main equipmentlist. One is a glass measuring cylinder with a plasticstopper which holds 100 ml. This is shown in Picture L.The other measuring cylinder is made of plastic andholds 1,000 ml or one litre. It is thus a litre-measuringcylinder. The graduated centrifuge tube in Picture K is auseful way to measure volumes from half a ml to 10 orsometimes 15 ml.Measuring cylinders and centrifuge tubes are not avery accurate way of measuring very small volumes offluid; so we often use special graduated glass tubes calledgraduated pipettes. The pipettes in FIGURE 5-2 are calledstraight graduated pipettes and are different from thePasteur pipettes that were described in Section 3.9.-There are four kinds of straight graduated pipettes in themain equipment list. Three are quite big pipettes and arefor measuring 10 ml. 5 ml, and 2 ml of fluid. They areshown in Pictures D, E, and F. The fourth pipette is usedfor measuring small volumes of blood----& ml, & ml, or $ml. It is shown in Picture G. It is not marked as & ml, hml, or & ml, but as 0.1 ml, 0.05 ml, and 0.02 ml. & ml isthe same as 0.1 ml. & ml is the same as 0.05 ml, and fa mlis the same as 0.02 ml. These decimals of a millilitreare like decimals of a gram. If you do not understandthem, turn back to Section 5.1. Fill the larger straightgraduated pipettes by putting the top end into yourmouth and sucking (taking in air) as shown in Picture R,FIGURE 5-3. Fill the blood pipette by sucking through arubber tube and mouthpiece as shown in’ FIGURE 7- 1.Measuring cylinders are easy to use. Fill them to thegraduation mark you want and then pour out the liquid.Pipettes are not so easy to use. You will see that thepipettes in Pictures D, E, and F have ‘0’ at the top andare marked at 10, 5, or 2 ml near the bottom. Severalother kinds of pipette are also made, and you may begiven them. Some have 10,5, or 2 ml at the top and 0 atthe bottom. Other kinds of pipette are used in dIrerentways, and the methods which follow may not work withthem. In most pipettes some of the volumes are writtenbeside the graduations. But other volumes are not written,and you have to work them out. To help you to dothis the bottom part of the lo-ml pipette has been drawnlarger in Picture H, and the meaning of the graduationmarks has been shown. The bottom part of the 5-mlpipette has been drawn larger in Picture I, and thebottom of the 2-ml pipette has been drawn larger inPicture J. A circle on the pipette shows the part of itwhich has been drawn larger. Look at your pipettescarefully with Figure 5-2 beside Jjou. If you can understandthe bottom graduation&. you will easily untierstandthe others.METHODUSING A GRADUATED PIPETTE. FIGURE 5-3Practise using your pipette with water.Look at the graduations on the pipette carefully. Makesure before you start that you know which graduationsyou are going to use.Put the blunt end in your mouth. Put the sharp endinto the liquid you are going to measure. This is shownin Picture Ft. (The person in this picture should be holdingthe pipette with his right hand in the positionshown.1 Suck the liquid up to just above the ‘0’ mark onthe pipette.Take the blunt end out of your mouth and quickly putyour index finger over it so that no air can get in. Theliquid will fall a little and then stop as shown in PictureS.Move your index finger a little. so that some air getsinto the pipette. The liquid will start falling back into thebottle. Let the liquid fall until it has got to ‘0’ at the topof the pipette as shown in Picture T. The top of theliquid in a pipette is never flat. It is always curved. Thiscurved top of the liquid is called the meniscus. Get thelower part of the meniscus opposite the graduation youwant, as shown in Pictures T, U, and V. If the meniscusfalls below ‘0’. start again.When you have got the leve! of the fluid to ‘0’. touchthe end of the pipette against the inner side of the bottlethat holds the liquid. This will let any drops of liquid onthe outside of the pipette drain away.

5 1 Weighing and Measuring.these are graduated pipettes, Pasteurpipettes are used differentlyput your index fingerover the top of thethe pipettehere while vou areFig. 5-3 Using a graduated pipetteTake the pipette to where you want to put the liquid.Move your index finger again just a little. Let the fluidfall to the graduation you want. The person using thelo-ml pipette in Picture U wanted to measure 4.3 ml.He has let the meniscus fall to 43 ml.Touch the end of the pipette against the side of whateveryou are putting the liquid into. This will let the lastdrop of liquid drain from the end of the pipette.Put any liquid you have not used back into the bottlewhere it came from.Don’t put the point of the pipette into any liquidexcept when you aie filling it up.The blood pipette, shown in Picture G, FIGURE 5-2, isused differently from the ordinary straight pipettes, Readhow to use it in Section ‘7.1. where the method of measuringthe haemoglobin is described.Pipettes easily get dirty, especially if they are notproperly looked after. Read about how to clean them inSection 3.12.Remember these things when you use pipettes.NEVER PIPETTE ANY SOLUTION OFCYANIDE. Cyanide is very poisonous and may killyou. Potassium cyanide is used in testing the urine forINH.Take great care in pipetting strong acids or alkalissuch as hydrochloric acid or ammonia, Even their fumesmay burn your mouth. Don’t pipette these rrnfil you arewell practised. If you can, measurP them irl some otherway. It is often possible to use a Pasteur pipette and agraduated centrifuge tube instead of a straight pipette.ALWAYS OPEN BOTTLES OF STRONG ACID ORAMMONIA WITH GREAT CARE. Don’t let themspill.Wash pipettes with water after you have used them.This is very important with blood pipettes. NEVERLEAVE BLOOD IN A PIPETTE. If you do it willbecome blocked.5.8 Percentages and ‘parts’As you have read, the word cent means 100. A percentagemeans the number of something that there is in ahundred of something else. 3% acid in alcohol meansthat there are 3 ml (or pints or cupfuls) of acid in 100 ml(or pints or cupfuls) of solution. You co&’ make this bymixing 3 ml of acid and 97 ml of alcohol. You couldNOT make it by mixing 3 ml of acid and 100 ml ofalcohol. The final volume of the solution of acid inalcohol must be 100 ml (not IO3 ml), The easiest way tomake this solution is to measure 3 ml of acid and put itinto something which can hold 100 ml. such as a measuringcylinder. Alcohol can then be added up to the markwhich measures 100 ml. In this way we add 97 ml ofalcohol and make a solution which contains 3 ml of acidin 100 ml of solution.Percentage solutions of solids in liquids are made inthe same way. In Section I. 18 you read that 0.85%sodium chloride (commo[\ salt) in water was calledphysiological saline or ‘salfine’. To make this solution wecan weigh O-85 g of sodium chloride and put it into aloo-ml measuring cylinder. We can then pour water intothe cylinder to exactly the lOO-ml graduation mark anddissolve the salt. There will then be 0.85 g of sodium

I’- >- Why and how we measure colour 1 5.9chloride in 100 ml of solution-we will have made a0.85% solution.Percentage solutions are easy to work out if you aremaking up 100 ml of a solution. Most of the solutions inthis book are made up in lOO-ml volumes. Othervolumes may not be so easy. For example, to make up 50ml of 3% acid alcohol we could put l+ ml of acid in 50ml of solution (50 is half 100; l+ is half 3).We have said that we could have 3% acid in alcoholby putting 3 ml (or pints or cupfuls) of acid in 100 ml (orpints or cupfuls) of solution (97 ml or pints or cupfuls ofalcohol being needed). This can also be written as 3 partsof acid in 100 parts of solution (97 parts of alcohol beingneeded). ‘Parts’ is only another way of saying any weightor volume we like. But, if we use parts, they must be thesame parts bJ7 weight or volume for eveqhing wemeasure. Parts are not used in this book, but you shouldknow what they are.You will often see the letters mg %. In this book theymean the number of milligrams of a substance in 100 mlof plasma. Thus a blood sugar of 150 mg % means thatthere are 150 milligrams of sugar in every 100 millilitresof the patient’s plasma.COLOUR5.9 Why and how we measure colour (FIGURE 5-4)In the first part of this chapter you read about measuringweight and volume. You had to understand this beforeyou could make any reagents. In the rest of this chapteryou will read about measuring colour. You will have tounderstand this before you can understand some of themethods that are described later in this book. But. first ofall, you must understand why we need to measurecolour.The blood of healthy people contains many differentchemical substances. In sick people there are sometimesmore or less of these substances than there should be. Itis often useful to measure how much more or how muchless of a substance there is. One of the things we measureis the red substance called haemoglobin (see Section 1.9).In the blood of a few patients there is more haemoglobinthan there should be. But many patients have less haemoglobinthan they should have. We say these patients areanaemic (an = without, aemia = blood). Sometimes wemeasure the sugar in the blood to see if there is too muchsugar or too little sugar. Sometimes we measure the ureain the blood to see if there is too much or too little urea.Haemoglobin, sugar, and urea are all measured withthe help of colour. So you must learn how we can usecolour to measure them.We will start by taking blood as an example. Ifsomething is coloured red like blood, and we put a fewdrops of it into a test tube of water, the water will go red.If we add more blood, the water will get more deeply red.If we could measure how deeply red the water was. wewould know how many drops of blood had been addedto it.Let us say, for example, that we have a tube of bloodand water. We do not know how many drops of bloodthere are in it and we want to find out. We call this tubeof blood and water our test solution-it is drawn inPicture A, FIGURE 5-4. What we could do is to takeseveral more tubes of water and put one drop of bloodinto the first tube, two drops of blood into the secondtube, three drops of blood into the third tube, and so on.These tubes have been drawn in Picture B and are calleda set of standards.We could then take our test solution and put it besideeach of the standard tubes in turn. We would find thatone of these standards had the same depth of redness asour test solution. The tubes of test solution and standardsolution have been drawn side by side in Picture C.When we find two coloured things are the same, we saythat they match one another. The two tubes in Picture Cmatch one another in colour.In this example we found that our test solution had thesame depth of redness as the fourth standard tube-thatis, the tube with four drops of blood in it. We can thereforesay that our test solution had four drops of blood init because it had the same depth of redness as the fourthstandard tube (it mntched the fourth standard tube).THE LOVIBOND COMPARATOR5.10 The Lovibond comparator (FIGURES 5-4 and 5-5)If possible&d a Lovibond disc and a Lovibond comparatorbefore you read this section. It is difficult to make aset of standard tubes each time we want to comparesomething, and it is easier to use pieces of coloured glassinstead. Instead of using tubes of a watery solution, weIIX coloured glass standard which are held in the windowsof a special black plastic disc (circle, ring). This setof glass standards in a plastic disc is called a Lovibonddisc and is drawn in Picture F. FIGURE 5-4. Lovibond isthe name of the man who first made this kind of disc.Each glass window in a Lovibond disc has a differentdepth of redness. just like our row of standard tubes.Unlike a set of tubes which cannot be kept for more thana few days, the glass standards of the Lovibond disc lastfor ever.The Lovibond disc is used inside the Lovibond comparator(comparer or something for comparing or matchingthings). The Lovibond comparator is a black plasticbox with two square holes in the top and four roundholes in the front. Look at Picture D. FIGURE 5-4, andPicture A, FIGURE 5-5. The four holes on the front areon a door which opens. Behind it is the Lovibond disc.The Lovibond disc sticks out at the edge of the door, andyou can turn it round with your fingers. If you turn thedisc you will see that the glass standards go round behindone of the holes in the door of the comparator. This is thestandard hole. Beside it is the test hole which is in frontof the empty place in the nG.Jdle of the Lovibond disc.The square holes in the top :bf the comparator are forspecial square tubes called Lokibond cells (ML 48d). one

-.. _--.--PROBLEMThis tube of water has some dropsof blood in it. How many dropsof blood are there? This is the I‘test solution’A-bloodi drops of blood bj 1x/ ‘4i 0d I LMETHODTake a row of tubes of water.Put one drop of blood into the3,first tube, two into the second4L;tube, three into the third tube SET OFand so on. This row of tubesSTANDARDS :is the ‘set of standards’r1\3\4’,5 6 72Put the test solution besideeach standard in turn untilyou find a standard thatmatches the testTEST SOLlJTlONthe tubes look the sameand are said to matchone another4th STANDARDANSWERBecause the test matches thefourth standard, the test musthave had four drops of bloodin itD ML13solutionTHE LOVIBONDCOMPARATORthe edge of theLowbond discsticks otttE Liquid standards. --a‘.. .. ,.:‘.I,::.’ a:..:- ;: ‘*‘ ‘p’: :._.,.)*.‘.::‘,>.:’._’II/l,,y ,_/ *‘-, ,_’_r /.,/AS;hese liquid standards in tubesare replaced by these glassstandards which last for ever -. ..-theHc‘\‘.i,/:answer holethe door opens sothat one dl$ccan be taken outand another putIn its placeSTANDARD,LUlturn the disc until you fir id a glass standardthat matches the test solu Ition exactly, thenread the answer from the answer holeIFig. 5-4 Why and how we measure colour

The Lovibond comparator 1 5.10THE LOVIBONO COMPARATOR OPENED USING THE LOVIBOND COMPARATORthe newer ‘Lovibond 100’/comparator uses squarecells.like thisLOVIBOND CELLA PLAN OF THECOMPARATORDML48elight lightwater 15I- P Itest tube for$ the older2kind ofcomparatori d 1/test solutionglass standard empty middle part of the discML48cIstandard hole ’ ’ “test hole \dooron the right hand and one on the left. When you shut thecomparator, you will see that the left-hand cell comesbehind the glass standards in the disc and the standardhole in the door. The right-hand cell comes behind theempty place in the middle of the disc and the test hole inthe door. At the back of the box are two round holescovered with white glass through which light gets intothe box.The comparator we have been describing is the newerkind called the ‘Lovibond 1,000’ comparator and hassquare glass cells. The older kind of comparator usesspecial rou& test tubes called Lovibond tubes (ML 48e).If you have the older kind of comparator, make sure thatyou ask for these found tubes to fit it. and not the newersquare cells.Now that we know about the parts of the Lovibondcomparator, let us see how we can use it to find out howmany drops of blood there are in a test solution. We startby putting the test solution into a Lovibond cell in theright-hand hole. Rettwtt~ber the test solrttiotl always goesinto the right-hard hole. In the left-hand hole we alwaysplace another Lovibond cell full of plain water. This ccl1Fig. 5-5 The Lovibond comparatorthis it is the part of the discthat you turn with your fingersof water is called the blank. The water blank and thecoloured glass standards work together in the same wayas the liquid standards in their tubes. Hold the comparatorup to the light and turn the Lovibond disc rounduntil you find one of the glass standards that has exactlythe same depth of redness as the test solution-that is.until you have found a standard which matches the testsolution. When you have found a glass standard thatmatches the test solution, read what the ‘answer’ isthrough the answer hole on the front of the comparator.Through the answer hole you will see a number oranswer written on the front of the disc. There is ananswer for each glass standard.Forget the fourth hole in the front of the comparator.it is not used with the methods in this book.Before we explain what the ‘answer’ means you mustremember that we have only been taking tubes of waterand drops of blood as an example. In our laboratory wedo not want to know the number of drops of blood thathave been added to a tube of water. IVe do want to klJOl1’how rmtch redress there is irl ow patient’s blood. becausethe redness is run& by the haerrtoglobirl in his blood. If

:, _ ,5 1 WeighingandMeasuring -’ ~~:’ “,’ ’ ” :we can measure the redness of a patient’s blood, we will Lovibond comparator after you have made the test solu-know how much haemoglobin there is in it. We do not tions for haemoglobin, sugar, or urea. These are deuseseveral drops of blood. We measure instead the red- scribed in Section 7.1 (haemoglobin), Section 7.42ness made by one drop of blood. If it is normal blood, it (sugar), and Section 7.4 1 (urea).will make a deeply red solution. If it is anaemic blood, itwill have little haemoglobin and only make a pale redsolution.METHODTo measure the haemoglobin we ta!ce a cell containingUSING THE LOVISOND COMPARATORa carefully measured amount (10 ml) of a special solution(haemoglobin diluting fluid), and add one carefully Put the test solution in the right-hand hole of themeasured drop of blood. This is our test solution. Wedon’t measure the blood as a drop. We measure it insteadcomparator.hand hole.Put a Lovibonddcell full of water in the leftinthe special glass tube called a blood pipette which you Hold the Lovibond comparator up to the sky or to aread about in Section 5.7 and which measures exactly & bright window to read it-look at Picture B in Figure 5-5.or O-05 of a ml of blood. The same amount of blood When trying to find a glass standard to match the testfrom different patients will contain different amounts of solution, put Your thumb over the answer hole and don’thaemoglobin. In healthy people 0.05 ml of blood will look at the answer until you have found a match, Youcontain plenty of haemoglobin, but in anaemic people it will then get the right answer, not the answer you thinkwill contain little haemoglobin. In healthy people the teat you want. When you have found an answer, try to get asolution will therefore be deeply red, but in anaemic match again, and see if You get the same answer. If youpeople it will be pale. We compare the redness of this test don’t, try a third time. You may have to give an averagesolution with the redness of the glass window standardson the Lovibond disc. When we find a glass standard thatexactly matches the test solution, we read the answer thatthis standard means front the numbers we see throughthe answer hole. These autllhcrs tell us how many gramsof haemoglobin the person has in every 100 ml of hisof several answers.blood-how many ‘g % of haemoglobin. This is what wewant to know about our patients. and this is the way inwhich their haemoglobin is reported. Measuring haemoglobinin blood has merely been taken as an example ofthe way in which we can measure things by their colourin the Lovibond comparator. The complete way to measurehaemoglobin is given in Section 7. I.It is easy to measure something red like haemoglobin.but how do we measure something which is white likesugar? What we do with colourless things is to make acolour with them. We add chemicals to a specimen ofblood so that the sugar in it makes a blue colour. Wemeasure the depth of this blue colour in the same waythat we measure the depth of a red haemoglobin solution.The more sugar there is in the blood, the deeper will bethe blueness. In the same kind of way we make a browncolour with the urea in the blood and measure the depthof the brown colour. The deeper the brown colour thegreater must be the-blood urea.We can use a Lovibond comparator to measure theblood urea and the blood sugar. There are two Lovibonddiscs with blue glass standards for measuring the bloodsugar. There are also two discs with brown glass standardsfor the blood urea. Two Lovibond discs are usedwith each of these methods, because there are not enoughglass stanr’ -ds in one disc alone. One disc in each pairhas glasc I: -idards for the lower amounts of sugar orurea. T; : ;I er disc has glass standards for the higheramounts 1,’ , dgar or urea. The Lovibond comparator isquite the, r) ; Id is the best machine for health centresand most o&patient departments.The following method tells you how to use theSometimes you wiii find that your test solution isdarker than one standard and paler than another. Forexample, it might be darker than the 5 9% standard andpaler than the 6 9% standard. When this happens givethe answer as half-way between them-5: 9%.Keep the Lovibond discs clean. They come in plasticboxes. As soon as you have finished using a disc, put itback in its box. You will not get a good match with adirty disc. If the glass standards get dirty, clean themwith a clean cloth.USE ONLY LOVIBOND TUBES; OTHER TUBESWILL GIVE THE WRONG ANSWER.THE GREY WEDGE PHOTOMETER5.11 The ‘Grey wedge’ (FIGURES 5-6 and 5-7)The full name for this instrument is the MRC (MedicalResearch Council) grey wedge photometer (light measurer).We will call it the ‘Grey wedge’. Look at it inPicture A. FIGURE 5-6.In the Lovibond comparator the test solution is put ina Lovibond cell or test tube. In the Grey wedge the testsolution is put in a glass ce!I. Look at these in Pictures Dand E, FIGURE 5-6. In the Lsvibond comparator thestandards are round pieces of coloured glass. In the Greywedge the standard is a grey ring or wedge. This is howthe Grey wedge gets its name. This grey wedge is on acircle of glass inside a metal wheel. Look for this wheelin Picture F, FIGURE 5-6. You can see the wheel as if itwere cut in half. In Picture B, FIGURE 5-7, this wheel hasbeen taken out of the instrument. You can see the glasscircle in the middle of the wheel. On the edge of the glasscircle you can see the wedge. One part of the wedge isnearly white, but, as you go round the ring, it gets darkerand darker until it gets nearly black. A wedge is somethingwhich is thin at one end and thick at the other.

The ‘Grey wedge’ 1 5.11The wedge here is an optical or light wedge. It is ‘thick tolight’ (very dark grey or opaque) at one end and ‘thin tolight’ (nearly clear or transparent) at the other end. Thedark end and the light end of the wedge have been bentround in a rine or circle until thev meet.Light goes lo the cell full of test solution through theclear middle part of the glass circle. Light goes to a cellfull of water through part of the grey ring. The light fromthe test solution and the light from the water are broughttogether by a prism and lenses (see Section 6.2). Youlook at this light through an eyepiece. The view throughthis eyepiece is shown in Pictures B and C in FIGURE 5-6.The light from the test solution is at the left of the fieldof view, and the light from the grey ring is at the right. InPicture B, FIGURE 5-6, the two halves of the field of viewthrough the eyepiece do not look the same: the right isdarker than the left. If you turn the wheel and lookthrough a different part of the grey ring, the two halvesof the field of view can be made to look the same. Thishas been drawn in Picture C. The wheel has been turnedso that a less dark part of the ring is behind the watercell. The right hand half of the field of view is lighter andis now the same as the left. Notice that the light from thetest solution goes to the left in the eyepiece, and fightfrom the wedge goes to the right. This is because lightcrosses over in the prism-look at Picture F, FIGURE 5-6.A A GENERAL VIEW OF THE GREY WEDGE PHOTOMETERcell f cell for water goes here ----1compartment 1 cell for test solution goes here ’at the scaleant to use your grey wedge bywithout electricity, unscrewnd hold it up to the lighton eyepieceGLASS CELLSFIELDOF VIEW,centre lineof prismlight notthe samein bothhalvesthe light is the same inboth halves : the halvesmatch one anotherML 115eGglassstandard/ulightfromwvringcell compartment,F A PLAN OF 7 ‘HE G ,REY WEDGE,darkside of the ringPHOTOMETERgrey glasslight.-over in the prismcell for test solution 1Fig. 5-6 The Grey wedge photometer

GREY WEDGEPHOTOMETERmetal wheelL grey ring(nearlyblack)the light from the test solution andthe light from the glass standardare made to match (look the same)by turning the Lovibond discIn the Lovibond comparator the meaning of each glassstandard (the ‘answer’) is written on each Lovibond disc.In the Grey wedge what each part of the wedge means iswritten on the edge of the wheel-look at Picture B,FIGURE 5-7. There are many Lovibond discs-one ortwo for each method. But there is only one wheel for theGrey wedge, and the numbers that are written on it arefor measuring the haemoglobin and the protein in theCSF. When the Grey wedge is used for measuring theblood sugar or the blood urea, the numbers on the wheelhave to be changed (converted). Ways of changing thesenumbers to give the answer for the blood sugar and theblood urea are given in FIGUKE 7-35. The numbers onthe wheel and the ways you are given to convert them areONLY for the methods given here. The numbers on thewheel will on& give you the right answer if you doEXACTLY what you are told in each ‘Method’.5.13, Filters for lightBlood is red, and the glass standards on the Lovibonddisc for measuring haemoglobin are also red. It is easytherefore to find a red glass standard which matches ared haemoglobin test solution. But the grey ring of theGrey wedge is grey. How can we match a red solutionand a grey ring?In trying to answer this you must understand that thewhite light from the sun that we see in the day (daylight)is a mixture of several colours. These are the colours ofthe rainbow or the colours of the spectrum. The rainbowis the curved line of colours that you sometimes see whenthe sun shines on rain as it falls from a cloud. White lightfrom the sun is split into colours by drops of water in thecloud. The colours of the rainbow or the spectrum arered, orange, yellow. green, blue and violet. All theseFig. 5-7’ The instrhments comparedtest solution / \ glass circlethe light from the test solution andthe wedge is made to match byturning the metal wheelrey ringIearly white)colours mixed together in the right amount make whitedaylight. If we look at a test solution of haemoglobin inwhite daylight, it looks red because red light gets throughthe solution. The other colours, yellow, orange, green,blue, and violet, have all been held up (stopped orabsorbed) by the haemoglobin solution. The morehaemoglobin there is in the solution (the greater theconcentration), the more will these other colours beabsorbed. We want to measure the haemoglobin in thetest solution; so we measure the light which is absorbed,not the light which goes through (is transmitted). Wemust therefore measure light of some other colour. notred. We choose green light and use a piece of green glasscalled a filter in the eyepiece of the Grey wedge. Look forthis eyepiece filter cap in Pictures A and F. FIGURE 5-6.A filter is called a filter because it lets light of only onecolour go through (in our example green). It holds backlight of the other colours of the spectrum (in our examplered, orange, yellow, blue, and violet). When we lookthrough the eyepiece, the view is green. When we turnthe wheel and match the two halves of the field of view,we are finding a piece of the grey ring which is going toabsorb the same amount of green light as the test solutionof haetnoglobin. Each place on the grey ring absorbsthe same amount of green light as a certain concentration(amount) of haemoglobin. This haemoglobin concentrationis written on the edge of the wheel.When measuring the haemoglobin the numbers on thewheel have been worked out for one kind of green lightonly. This is the No. 2 green eyepiece filter. This filter isalso used with a method given here for measuring proteinsin the CSF. Two other eyepiece filters are alsoprovided with the Grey wedge-thev screw into theinside of the lid of the box that holds *it. One is the redNo. 1 filter which is used for the blood sugar. The otheris the No. 3 filter which is a different kind of green and is

Ii’ used for the blood urea. THE RIGHT FILTER MUSTBE USED FOR EACH METHOD. THE WRONGFILTER WILL GIVE YOU THE WRONG ANSWER.This is also true for the EEL calorimeter.5.13 The Haldane scaleThe L&bond haemoglobm disc (Number 5/37X) hasthe ‘answers’ 3, 4, 5, 6, 7, 8, 10, 12, and 14 for its nineglass standards. These are the number of grams ofhaemoglobin in 100 ml of blood (g%). But the wheel ofthe Grey wedge has numbers from 0 to 260 on it. Whatdo they measure? The numbers on the wheel of the Greywedge measure the patient’s haemoglobin as a percentageof normal. This is called the Haldane scale. Normal onthis scale is 14.6 g%; so a person who is 100% normalhas 14-6 g of haemoglobin in 100 ml of his blood. Thepercentage way of measuring haemoglobin is an olderway of doing it, and most people now use g%. There is ascale in FIGURE 7- 1 with which you can change percentageon the Haldane scale into g%-or g% into pcrcentageHaldane.5.14 METHODUSING THE MRC GREY WEDGE PHOTOMETERFOCUSING ON THE CENTRE LINETurn on the light in your Grey wedge. Take out thecells from the cell compartment and turn the wheel to‘lo’.Look at Picture A in Figure 5-6 and find the ‘knurledring on eyepiece’. Turn the knurled ring on the eyepieceone way and then the other. You will see the line in themiddle of the field sharply (it will be ‘in focus’-eeSection 6.7). As you turn the knurled ring more, the linein the middle of the field of view will get hard to seeagain (it will go ‘out of focus’). Turn the eyepiece oneway and the other until the centre line is sharply infocus. WHENEVER YOU USE THE GREY WEDGE,MAKE SURE THAT THE EYEPIECE IS FOCUSED ONTHE CENTRE LINE IN THE MIDDLE OFTHE FIELD OFVIEW.CHECKING THE READINGS ON THE WHEELWith every Grey wedge there is a little block of woodwith a hole in it. This is the standard block and is shownin Picture G, Figure 5-6. Inside the hole is a little circleof grey glass. This grey glass is a standard to help makesure your Grey wedge is accurate. On the block is apercentage figure. In the block that has been drawn inPicture G this is 96%. but your block may be different. Ifthe Grey wedge is accurate this standard should alwaysread 96%. Put your standard in the right-hand cellcompa,rtment. LEAVE THE LEFT-HAND CELLCOMPARTMENT EMPTY. Use the No. 2 eyepiece. Takethe average of several answers. Is this average answerthe same as that on the grey glass standard? If it is notthe spme; always add on or take away the differencefrom every answer you get. If your average is 9696, youare reading 2% too high. Take away 2% from all yourhaemoglobin answers. If, for example, you read 37%.report 35%; if you read 62%. report 60%. If you shouldget ari answer of 96% with your standard block and youget au average answer of 96%. add 2% to every answeryou get.MAKINGA READINGFill the left-hand cell with water. If possible usedistilled water-see Section 3.15.Put the test solution in the right-hand cell. MAKESURE YOU ARE USING THE RIGHT FILTER(Haemoglobin No. 2, CSF protein No. 2, sugar No. 1,urea No. 3).Look through the eyepiece. Match the right and lefthalves of the field of view by turning the wheel withsmaller and smaller movements until both halves lookexactly the same. Where possible take the average ofseveral answers. Maka some readings by starting withthe right hand of the field too bright and others with ittoo pale. If, for instance, you get the answers 64.63.65,66, 67, report the average which is 65 (see Section 1.3).Don’t look at the scale until afier you have made yourreading-this will help you to get the true answer andnot the answer you think you should have.SOME FURTHER POINTSKEEP THE CELLS CLEAN. If cells get dirty, cleanthem inside with a swab of cotton wool on the end of anapplicator stick.Hold a cell by its sides. This will help to keep its backand front clean.ALWAYS PUT THE CELLS INTO THE CELLCOMPARTMENT WITH THEIR OUTSIDES DRY.Keep the glass windows at the back and front of thecell dompartment clean. If these get dirty, you will getthe wrong answer. The best way to stop them gettingdirty ‘is to put only clean cells into the cell compartment.To see these glass windows, look into the cell compatimentfrom the top. They are shown in Picture F. Figure5-6. A:8 they clean?Make sure that the cells are always nearly full, especiallythe water cell on the lef?. Light must go throughwater or solution in the cell, and not through air.WASH AND DRY THE CELLS AS SOON AS YOUHAVE FINISHED WITH THEM. Never leave a cell fullof test solution in the cell compartment of the Greywedge.Put your Grey wedge on a shelf, or on a box on thebench at a height that is easy for you to use when sittingdown.If you have not got electricity, use daylight. Unscrewthe lamp house and hold the Grey wedge up to abrightly lit window.Keep your Grey wedge in its box away from the dust,or make it a cover of plastic sheet to keep the dustOff.

i 1 Weighing and MeasuringA AN OUTSIDE VIEW , pointerB AFILTERfilternumber.cAN EEL . TUBEML 117~[irough ground linemake sure thisrough ground lineis opposite the‘wsition line’1 dn tlye.EEL ,hfn1 You ratce a reactmg/when the on and off switch is‘off’, the point of thegalvanometer is held still,and the galvanometer is lesseasily harmed by jolts, soalways keep this switchoff when not using the EEL,and always keep it offwhen the EEL is movedD HOW THE EEL WORKSE THE GALVANOMETER SCALE%this is themark forinfinityshutterpointer set at ‘0’. ---much light isfalling on theSelenium cell’ wires to galvanometerwhen light shines on the seleniumcell the pointer moves to the leftpointerat rest,no lightIS shining3n theseleniumcellHHOLDER FORF SMALL EEL TUBESNEUTRALG STANDARDGREYAMPOULE OFCYANMET-HAEMOGLOBINstude-EELtubekeep these standardsin the refrigeratorand use a new ampouleeach daytube filledwith greysolutionthis standard is ------only for measuringhaemoglobinFig. 5-8The EEL calorimeterML 127a

-Measuring colour with electricity 1 5.16THE EEL COLORIMETER5.16 Measuring colour with electricity (FIGURE 5-8)With an EEL calorimeter (colour measurer) we can measurethr depth of the colour of any coloured solution.The measuring is done with electricity, and there is nothingto be matched by eye (unlike the Lovibond comparatoror the Grey wedge). The instrument is called theEEL calorimeter after the people who make it (EvansElectroselenium Limited). We will call it ‘the EEL’. TheLovibond comparator cannot go wrong. The Grey wedgeseldom goes wrong, but the EEL does sometimes gowrong. This is one of the difficulties with the EEL, bot ifyou h::ve spare parts, you may be able to mend it. Whenthe EEL is working properly, it is quick and easy to useand is more accurate than either the Lovibond or theGrey wedge.The important part of an EEL is a flat piece of metalcalled a selenium cell-look at Picture D, FIGURE 5-8.(This is yet another use of the word ‘cell’.) When lightshines on a selenium cell, some of it is made into electricity.The more light that shines on the selenium cell, themore electricity will it make. When it is dark the seleniumcell makes no electricity. Electricity from the seleniumcell goes along two wires to a machine called agalvanometer. The galvanometer measures electricity. Ithas a hand or pointer and a curved line of numbers andgraduations called the scale-look at Picture E, FIGURE5-8. When no light fails on the selenium cell. it makes noelectricity, and the pointer stays resting at the right-handside of the scale: this is marked infinity or ‘c1c’. But whenlight falls on the selenium ccl! it makes electricity. Thiselectricity goes to the galvanometer and makes thepointer move towards ‘0’ at the left of the scale. With aselenium cell and a galvanometer we can thereforemeasure light. But how do we use them to measure thedepth of a coloured solution?Inside the EEL light comes from a bulb like the bulbof an electric torch-look at Picture D. Light from thebulb goes between two pieces of metal called the shutters.These shutters can be opened and closed like the shutterson a window. Between the shutters is an empty placecalled the slit. By moving the shutters we can make theslit big or small. The shutters can be opened and closedby turning the increase light wheel on the top of the EEL.Turning the increase light wheel to the right opens theshutters wide and makes the slit wide. Much light will gothrough the slit to the selenium cell. Wheu the shuttersare nearly closed (the increase light wheel turned to theleft! the slit will be narrow. and littl,: light will gothrough. Light from the slit goes through a special testtube called an EEL tube. Light which hzi gone throughthe EEL tube then goes through a colsured glass filterlike that 03 the eyepiece cap of the Grey wedge (seeSection 5.12). This filter lets light oi’ onl:/ one colour fallon the selenium cell. The selenium cell turns thiscoloured light into electricity which moves the pointeracross the scale of the galvanometer.Now that you know something about the EEL, youcan learn more about how it works. First, you must get itready to use.6.17 METHODGElTlNG THE EEL READYUnpack your EEL carefully. You will see a ‘Warningnotice’ under the glass of the galvanometer. Take thisout of the EEL by unscrewing the ‘screws to fix cover’(Picture A, Figure 6-8). Read this notice carefully.USlNG THE EEL WITH MAINS ELECTRICITYYOU will see a large thick wire coming out of the EEL.Inside it are three small wires: a green and yellow wire,a blue wire, and a brown wire. Join the green and yellow‘earth wire’ to the big brass (metal) pin at the end of theplug. The blue and the brown wires can go to either ofthe other two pins. See that the mains/battery switch onthe right at the front of the EEL is turned to ‘mains’.USING THE EEL WITH A BATTERYFix two wires to each of the two screw terminals (aterminal is something an electric wire is fixed to) at theback of the EEL. Fix the other ends of these two wires toa two volt batmy. You can use a 2-volt dry battery orone cell of a car battery (used like this a cell is one of theparts of which a battery is made).It is also possible to use a special battery for the EEL(ML 117k) which can be charged with a trickle charger(ML II7j) from the mains-see Section 3.8. Two kindsof battery can be recharged in this way. One is the leadacidkind which is filled with dilute sulphuric acid andmust be kept charged and topped up with water, just likea car battery. The other kind is a nickel-iron alkalibattery, which is filled with caustic soda (sodium hydroxide)solution. This will not spoil if it is not keptcharged. Fill whichever kind of battery you have with theright solution, and use it the way the instructions say.Now that you have got your EEL ready, you can seehow it works.5.18 METHODLEARNING HOW THE EEL WORKSPut the green filter llford No. 625 into the EEL. llfordis the maker’s name. Open the shutters wide by turningthe increase light wheel to the right. Turn on the lightand put a tuba of plain water into the EEL. This tube ofplain water is called the b/ank tube. Plenty of light willnow go through the wide open shutters, through theblank tube, and fall on the selenium cell. The seleniunicell will make plenty of electricity, and the pointer of thegalvanometer will go rapidly beyond ‘0’ at the left of thescale.Close the shutters a little by turning the increase light

5 1 Weighing and Measuringwheel to the left. The pointer will move to the right.Move the increase light wheel just enough for the pointerto come to ‘0’ (this is shown by the dotted line inPicture E, Figure 5-8). Like this the EEL is ready to uselighton. tube of wafer in place. filfer in place. and theincrease light wheel turned to bring the pointer to ‘0’ onthe scale.Take out the blank tube of water. Put a weak testsolution of haemoglobin into the EEL: the pointer willleave the ‘0’ mark and stop somewhere on the scale, sayat 2. The dilute solution has absorbed (taken up orstopped) some of the light and too little of it nowreaches the selenium ceil to make enough electricity topush the needle to ‘0’. Take out the pale coloured solutionand put back the blank tube of water: enough lightwill again reach the selenium cell to send the needle to‘0’.Next put a strong solution of haemoglobin into theEEL. This solution will absorb more light than the dilutesolution. Less light will fall on the selenium cell; it willmake less electricity, and the needle will stop even furtherto the right at, let us say, 5 on the scale. Therefore,fhe higher the scale reading. the greater must be thedepth of colour of a haemoglobin solution, and the morehaemoglobin there must be in if (fhe more concentratedit must be). With the EEL calorimeter we can measurethe concentration of any coloured solution, not onlyhaemoglobin.The numbers on the scale. from ‘0’ at the left to 10 onthe right, are placed to give a true measurement of theamount or concentration of the substances we are measuring.The numbers are far apart near ‘0’ on the left andclose together near ‘10’ on the right. This kind of scale isdifferent from the scale on a ruler where all the numbersare the same distance apart. A ruler has a ‘linear’ scale;the EEL has a ‘logarithmic’ scale.5.19 Standards for the EELIn the Lovibond comparator the ‘answer’ is written onthe Lovibond disc. In the Grey wedge photometer the‘answer’ is written on the edge of the wheel. These instrumentsare easy to use because they have standards insidethem with which the test solution can be compared. Inthe Lovibond comparator these standards are thecoloured glass windows on the Lovibond disc. In theGrey wedge the grey ring or wedge is the standard. Butthe EEL has no standard inside it. The EEL can only tellus that one solution is more deeply coloured than anotherand thus has more of a coloured substance in it. TheEEL can also tell us exactly how much more: a solutionfor which the pointer reads 40 has twice as much of asubstance in it as one for which the pointer only reads20. But the EEL cannot by itself tell us what the figure20, for example, means in grams percent of haemoglobin.Before we can use the EEL with any method we musthave a standard whcse value we know. It is not possibleto use the EEL without a standard. The best standard touse for measuring haemoglobin is a solution of a kind ofhaemoglobin called cyanmethaemaglobin (ML 127a,Picture II. FIGURE 5-8). In the same way it is possible touse standard solutions of sugar and urea when measuringthe blood sugar and blood urea. But all these standardshave difficulties and none of them keep well. We havethus to use another kind of standard called a neutral greystandard (ML J171, Picture G, FIGURE 5-S). This is agrey chemical solution which is sealed into an EEL tubeand can be kept for many years. This neutral greystandard can be used for measuring the haemoglobin, theblood sugar, or the blood urea. The cyanmethaemoglobinstandard can, however, only be used for measuring thehaemoglobin.The neutral grey standard is grey, not red like haemoglobin,but this does not matter because its job is to letthrough a standard amount of green light when used witha green filter. This green light is equal to that whichwould get through a tube of standard haemoglobin if wewere to use one. All the methods for measuring haemoglobinon the EEL use the same green Ilford 625 filter.Once you have understood how to use the EEL withone method it is easy to use it for other methods. Weshall describe the measurement of haemoglobin as anexample. What haemoglobin is and how the specimen istaken is described in Section 7.1.All the methods for measuring haemoglobin in thisbook use 0.05 ml of blood added to 10 ml of solution.The blood and solution together measure 10.05 ml. Thismakes a dilution of one in 20 1 (IO.05 + 0.05 = 201).We usually forget the ‘one’ and say we have a dilution of‘one in two hundred’.5.20 The cyanmethaemoglobin methodIt is not easy to use the ordinary haemoglobin of bloodfor a standard for the EEL because it does not keep well.But, when haemoglobin is changed into cyanmethaemoglobin,it keeps better and can be used as a standard.These cyanmethaemoglobin standards are boughtalready diluted and sealed into ampoules, each box ofampoules having a slightly different value. Theseampoules are labelled with their haemoglobin values inmilligrams of haemoglobin per 100 ml. Most standardsare about 60 mg per 100 ml.0.05 ml of the patient’s blood is taken into 10 ml of aspecial solution called Drabkin’s solution (Section3.3 lb). Drabkin’s solution contains a little potassiumc-1,.anide and some potassium ferricyanide. It can be madeup from chemicals or from special tablets (ML 127b).Drabkin’s soluticin changes the haemoglobin in thepatient’s blood into cyanmethaemoglobin. The cyanmethaemoglobinin the test solution is then comparedwith the standard.The cyanmethaemoglobin method is the most accurateone for measuring haemoglobin but has several diffculties.One of them is that, once an ampoule of standardhas been opened, it only keeps for a day. At the end of aday it must be thrown away. The unopened ampoulesonly keep for a few months and have to be stored in a

Oxyhaemoglobin methods 1 5.2T arefrigerator. Also, Drabkin’s solution is made with cyanide,and, although it contains so little cyanide *hat it isnot dangerous by itself. the solid cyanide from which it ismade is very dangerous indeed (see Section 8.9).METHODMEASURING HAEMOGLOBIN USING THE CYAN METHAEMO-GLOBIN STANDARDMake Drabkin‘s solution either by dissolving one ofthe special tablets in water. or by the method describedin Section 3.31 b.Measure lO-ml volumes of Drabkin’s solution intouniversal containers or EEL tubes.Add Q 05 ml of the patient’s blood to the Drabkin’ssolution, following the method described in Section 7.1.Mix well and let the mixfure stand for fen minutes.During this time the red cells will lyse, and the cyanideand ferricyanide in Drabkin’s solution will make thepatient’s haemoglobin into cyanmethaemoglobin.Take an ampoule of standard from the refrigeratorand let it get warm in the room for ten minutes. Make asmall scratch on the neck of the ampoule with a file.Break open the ampoule and pour it into an EEL tube.This is your standard solution.Put a green I!ford 625 filter into the EELPut an EEL tube of plain Drabkin’s solution into theEEL This tube is called the ‘blank’. Adjust the increaselight wheel to bring the pointer to ‘0’ with the ‘blank’ inplace.Quickly take out the blank tube and put in the standard.Take a reading. Put the blank back and bring thepointer to ‘0’ again.Put back the standard-Do you get the same reading7If you do, go on to the next step. If you do not, take theaverage of several readings (see Section 1.3).Do exactly the same thing with the test solution-setthe pointer to ‘0’ with the blank tube of Drabkin’s soiution,and take the average of several readings if necessary.You will now have a reading for your standard solutionand a reading for your test solution. Work out theanswer in the following way:Pat&t’s haemoglobin in g %Test reading x Figure= Standard readingon ampouleSay your standard reads 40, your test reads 20, and thefigure on the ampoule reads 59.8 mg of haemoglobinper 100 ml. Your calculation would be:20 59.8- x - = 5.98 g %, say 6 g %, of haemoglobin.40 5We get the figure ‘5’ in the working out above likethis:Haemoglobin value Haemoglobin value on theof the standard=ampoule in milligrams %in grams ?41 1,0005x 20120 1 can be divided into 1,000 very nearly five times. 20 1is what is called the ‘dilution factor’ and is the number 0%’times that you are going to dilute the blood to make thetest solution. If you are using other blood pipettes thanthe O-05-ml pipette we describe here and volumes ofDrabkin’s solution other than 10 ml, you will have towork out a different dilution factor.Answers are more easily worked out with the helpof the graph described below. To make this graph youwill need the value of the standard. Get this by dividingthe haemoglobin value in mg % found on the ampouleby 5. It will probably be direrent with each box ofampouies.5.21 a Oxyhaemoglobin methodsIn these methods 0.05 ml of blood is taken into 10 ml ofdilute sodium carbonate or ammonia solution. Thesechemicals lyse the red cells and let out their haemoglobininto the solution. Oxygen (a gas from the air) turns thehaernoglobin into oxyhaemoglobin. This oxyhaemoglobinis usually just called ‘haemoglobin’ and can bemeasured with the Lovibond comparator, the Greywedge, or the EEL (see Section 7.1). ‘*METHODMEASURING OXYHAEMOGLOBlN USING THE EEL AND A NEUTRAL GREY STANDARDMake your test solution from 0.05 ml of blood and 10ml of sodium carbonate or ammonia made as describedin Section 3.31.Take readings of the standard and the test solutionsexactly as described above, using an llford 625 filter anda water blankPatient’sTest readinghaemoglobin =in g %Standard readingx 14.6This will be easier to work out if you use the graphdescribed in the next section.5.21 b Using a graphIt wastes time to do a sum every time we measure apatient’s haemoglobin, and it is easier to use a graph likethat in FIGURE 5-9. A graph is a special picture for doingarithmetic. This graph is drawn on squared paper, but incase you have not got any squared paper,, FIGURE 5-10has been drawn for you. It is at the end of the book withthe other figures you can tear out. There is nothing onthe back of this Figure, so that you can cut it out of thisbook and use it in your own laboratory. FIGURE 5- 10 israther small, and it is better to find a larger piece ofgraph paper and uce that. There are more accurate waysof making a graph for the EEL, but this is the easiestway.

welgnmgana measumgllford filter 6 25, one part ofwith the EEL calorimeter that wasblood diluted in 200 parts of waterused to make this graph the neutralgrey standardiIgave a reading3 ! ,I I I II ,,I I, I, “I nf 72 on the SC :aleI I I I ! I II I i I i I1 I I I I I if&i 1NEUTRALSTANDARDEEL TUBEGREYIN ANML 117 II!Y!!!!r!!!!‘!llllyl ! I ’ I j I’II ’ II’ I I I I I ! I 1‘read ihe’bottdrrI0 12 3456789’ 10 'II 12 13 14 t 15 16HAEMOGLOEIN IN GRAMS PER 100 ML. - ‘GRAMS %’ 14.6the neutral greystandardis equivalent(equal) to14.6 g % ofhaemoglobinby the methoddescribed hereFig. 5-9A graph for the EELMETHODMAKINGA GRAPHTake a piece of squared paper. Make a scale along thebottom to show the haemoglobin in grams from 0 to 16.Make a scale up the side from 0 to 6 for the EEL reading.The cyanmethaemoglobin standard, and the liquidgrey standard can both be used to make a graph. Let ussay, for example, that you are using the liquid greystandard which is equal to 146 g % of haemoglobin.Using the llford 625 filter, you find perhaps that thisstandard gives a reading of 72 on the scale of the EEL.Find the 14.6 line on the bottom scale of the graph andfind the 72 line on the scale at the side. Make a point (adot) called S (S for standard) at the place where theselines cross. Join 8 to 0 on the scale.Using this graph is very easy. Let us say your testsolution gives a reading of 35 on the EEL. Find where 35comes on the scale at the side. Go along to the 35 lineuntil you get to the line from 0 to 6. When you come tothis line go down to the bottom scale. This will give youthe haemoglobin in your test solution in g %.Used like this your graph (FIGURE 5- 10) will soon getdirty. It is better to get an old X-ray film and wash off theemulsion with hot water-the emulsion is the ‘paint’ onthe film that makes the X-ray picture. The film will goclear. Cut a piece of cardboard and a piece of film thesame size as FIGURE 5 10. Put the figure between thecardboard and the film and put sticking plaster roundthe edge. Mark point S with grease pencil. Draw the linefrom S to 0 with grease pencil. Better still, get a strip ofX-ray film about one inch wide. Scratch a line on it anduse this instead of grease pencil as your line from 0 to S.

When an EEL goes wrong 1 5.22This is narrower than a grease pencil line, and you canmove it if the place of y+Jur S moves. Fix the strip whereyou want it with surgical tape or drawing pins.The readiig given by a standard may change, so,CHECK THE READING GIVEN BY ANYSTANDARD YOU USE OFTEN-ATLEASTEVERYDA Y. If it changes, move poim S on your graph, and joina new line from 0 to S. This is another reason why it isbetter to cover FIGURE 5 - 10 with X-ray film and use astrip of film with a line on it for the line from 0 to S.METHODMAKING THE BEST OF YOUR EELDon’t keep your EEL on the same bench as a centrifuge.The continual shaking of the bench will harm-theEEL. Keep it on a bench where it will not be shaken. Ifthis is not possible, keep it on a soft thick rubber mat, oron several layers of blanketBefore you use an EEL, switch it on for about 5 minutesand leave it with the shutters closed (the increaselight wheel turned as far as it will go to the left). An EELis more accurate if it has been warmed up like this.Make sure that there is enough test solution or waterin an EEL tube to cover the path of the light goingthrough it This means that EEL tubes must be at leasttwo-thirds full.Keel., the EEL switched on all the time when severaltests are being done. Don’t swirch it on and off eachtime a tube is put into it. Do switch it off if you are nogoingto use it for more than about 15 minutes. Don’tleave the EEL on when you go for lunch!After every reading with a test or standard solution,always make sure that the pointer still reads ‘0’ whenthe ‘blank’ tube is put back into the EELRead the place where the needle comes to restrapidly. Don’t spend too long watching the needlebefore taking a reading.Close the light cover (Picture A, Figure 5-8) whentaking a reading. This stops light from the room gettingto the selenium cell.Keep the light cover closed when the EEL is not beingused.EEL tubes have a rough (ground) mark on them. Lookat Picture C in Figure 5-8. Turn an EEL tube so that thisground glass line always comes opposite the ‘positionline’ on the top of the EEL Look at Picture A, whichshows this. You will get a more accurate answer if youalways keep an EEL tube turned to the position line likethis.Make sure the EEL tubes are clean and that there areno bubbles on the sides of the tube when you take areading. Hold tubes by their tops, and put them into theEEL dry.Unless you are using the EEL. keep the ON-OFFswitch turned OFF. This protects the moving parts ofthe galvanometer. Don’t put the needle to the rightmerely by closing the shutters. ALWAYS CARRY ORTRANSPORT AN EELWITH THE SWITCH TURNEDOFF.THE EEL IS AN EXPENSIVE INSTRUMENT ANDEASILY BREAKS. LOOK AFTER IT CAREFULLY.5.22 When an EEL goes wrongYou will need some spare parts or spares. The mostimportant of these are spare bulbs. There are specialclips (holders) to hold these spare bulbs inside the EELat the back of the galvanometer. Some bulbs give muchmore light than others. You should also keep a spareselenium ce!l because you will probably have to put anew selenium cell in your EEL about once every 18months. Make sure your spare cell is kept well wrappedup in a dark place. It is also useful to keep a sparegalvanometer. But this is expensive, and there is no needfor every laboratory to have one. If there are three orfour in a medical Store or Central Laboratory, they canbe used as they are needed. Galvanometers which do notwork can usually be mended by the makers.Here are some of the things that can go wrong with anEEL.The pointer does not move when the r’ight is switched on.Is any light coming from the bulb? Look down thehole in the top of the EEL and see.1. There is no tight.(a) Is the EEL plugged into the mains? Or joined tothe battery?(6) Is the bulb loose? Screw it in (see Section 5.23).(c) Has the bulb broken? Put in a new bulb (seeSection 5.24).(d) Has the fuse in the mains plug fused (broken)?Put in a new fuse.(e) Is the ‘mains-battery switch’ rightly switched?2. There is light.(a) Is the stud (a stud is a short rod) of the tubeholder in its slot (hole)? If it is not, the tube holdermay be turned round and may ,be blocking the path ofthe light from the bulb to the!ielenium cell. See thatthe rod is in the slot. A tube holder is used for holdingsmaller sized tubes, and is shower in Picture F, FIGURE5-8.(b) Are the ‘contacts for the selenium cell’ loose?Tighten them up (see Section 5.25).(c) Is the selenium cell working? Put in a new seleniumcell (see Section 5.25).The pointer does not reach ‘0’ with a blank EEL tube ofwater in place, or the pointer moves very slowly.(a) Is the EEL tube full of water? Fill it up.(b) Is the filter clean? Clean it.(c) Is the reflector clean? Clean it with a sofS cloth.(d) Is the stud of the tube holder in its slot? See thatit is.

5 1 Weighing and Measuring(e) The bulb may not be giving enough light. Tryusing a new one.cf) Is the selenium cell working? See if you canfocus the light better (see Section 5.23). If this doesnot work, change the selenium cell (see Section 5.25).The pointer does not move back to infinity (Picture E.Figure 5-S).The pointer should always be at CC when the switch isOK. the light cover closed. and the EEL not joined to themains or the battery. If the pointer is not at CC, it needsadjusting. Look at FIGURE 5- Il. Turn the infinity adjustingscrew until the pointer comes to infinity. If the pointersticks and does not come to infinity, change the galvanometer.The EEL will no: -xork accurately unless the pointercomes back to infinity.The EEL sometimes works well and somei&zev does nos.The wires and the terminal screws of the bulb, seleniumcell, or battery may be loose. Tighten them up, and ifnecessary change the bulb or the selenium cell.The pointer sticks, stops, or moves in jerks.The galvanometer is not working. Try very gentlytapping the infinity adjusting screw with your fingers.T’he pointer may become free. If the pointer does notbecome free., change the galvanometer.Here are some other things you can do to meIEEL.You never seem able to get the same reading twining.ILf you have a mains EEL it may be because the(strength) of the mains electricity keeps changing.a common cause of trouble, and the’only way toit is to use a battery. You can use iI large 2.5-kbattery. Even better is an accumulator (ML 117ktrickle charger (ML 117j). An accumulator storltricity and can be charged (filled up) from the maithe trickle charger. This is described in Section 3mains electricity may alter but the electricity frbattery will be constant (the same) and will givtsteady reading.Another reason may be that there is a loosesomewhere. So, see that the lamp is firmly screthat the contacts for the selenium cell are not lotthat the wires to the mains or battery hqe not corn5.23 METHODFOCUSING THE BULB OF THE EEL, FIGURE 5-11Unscrew the knobs holding the cover andcover off. Turn on the light; put in the filter you \igalvanometerthis knob holdsthe coverbulb holder,--k-thiskmthe covmains/t/------ $y;;reflector inside A/ slot of therebulb-ML 1179-t seleniurtermina-2-seleniumcellcover‘l place fcEEL tushuttewheelFig. 5-l 1 Changing a bulb on the EEL

When an EEL goes wrong 1 5.22BEFORE STARTING ;selenium cell terminal scre&WITH THE CELL COtiERREMOVEDj ._., N,:.‘;..-.I , 5 ,t .IWITH THE SELENIUM CELL REMOVEDTHE SELENIUM CELL TAKEN OUTD ML 177hNi selenium cell 1bright andshiningrough and dullLEFT ’RIGHTuse and also a tube of water. Turn the wheel as far as itwill go to the left to open the slit. Loosen the ‘bulbholder fixing screw’ and move the bulb holder in and outof the ‘slot’ shown in the figure until the pointer is furthestto the left. There will be one place in the ‘slot’where the bulb will be focused on the selenium cell aridwhere the pointer will move furthest to the left. Whenyou have found this place, tighten up the bulb holderfixing screw.5.24 METHODCHANGING THE BULB IN THE EEL. FIGURE BllFig. 5-12 Changing a selenium cell on the EEL5.25 METHODCHANGING THE SELENIUM CELL OF THE EEL. FIGURE 112Take off the cover of the EEL as described above. Findthe selenium cell cover and see which coloured wire isgoing to the terminal screw on the right and which tothe terminal screw on the left. Remember these colours.Unscrew the ‘selenium cell terminal screws’. Take thewires, nuts, and washers off the ‘selenium cell terminalscrews-. Take off the ‘selenium cell cover”Picture B.Loosen the nut X’ on the ‘rear contact’. Move the ‘rearcontact’ downwards, as shown by the arrow in PictureC. Take out the selenium cell.Unscrew the knobs holding the cover and lift it off. Put in a new selenium cell---the shining front sideLoosen the bulb holder fixing screw and gently pull oyt must be next to the glass. Put back the rear contact andthe bulb holder as shown. Put in a new bulb. Screw it tighten up the nut ‘X’. The selenium cell must be tightlytight. If this is your last bulb, order some more. Put back held between the contacts. You may have to bend thethe bulb holder. Tighten up the bulb holder fixing screw rear contact so that the selenium cell is tightly held. Putand put back the cover. If necessary, focus the bulb as back the selenium cell cover and the nuts and washersdescribed in the method above. of the selenium cell terminal screws. Put the wires back

5 1 Weighing and Measuringexactly as you found them. First there is the cover, thena washer. then a nut then the wires, and then, last of all,the second nut Tighten the nuts4ut not too much oryou may break the cover. Put back the cover.QUESTIONS1. Give some examples of fractions and decimals of agram.2. Name and draw diagrams of the various pieces ofequipment that can be used to measure the volumes ofliquids.3. Describe the way in which we can use colourto measure the concentration of a substance in asolution.4. What must you do to get accurate readings with anEEL? In what ways can an EEL go wrong, and’how canit be put right?5. How would you use the Ohaus balance to weigh 3 1 gof sodium carbonate?6. Why do we measure ‘depth of colour’ in a medicallaboratory?7. Describe the Lovibond comparator and the way itworks.8. What must you do to make sure you get an accuratereading with your Grey wedge photometer?9. What is a filter? Why are filters used on the Greywedge photometer and the EEL calorimeter?10. What are the advantages and disadvantages of theliquid grey standard that can be used with an EEL colorimeter?

6 1 The Microscope6.1 The pm your microscope, you should read this chapter very carefullyseveral times. Most of the pictures are of theThe microscope is the most important machine in a Olympus Model K microscope, which is shown inmedical laboratory: it is often the most expensive FIGURE 6-1. But most of what is said in this chapter ismachine also. If you are going to make the best use of true for all microscopes. Olympus is the name of theeyepieceML 30bthe whole tube can moveround in a circlerevolvingnosepiece withthere are only threeobjectives in place,the place for thethird is closed bya caphold the microscopewhen you carry itherelow power objectivearmML 30amirror --coarse adjustmentfine adjustmentcondenser adjustingknobyou are looking at the microscopefrom the left hand side ; make sureYOU put your own microscope thesame way round as this pic?urebaseNote: There is nomechanical stage onthis microscopeFig. 6-l The Olympus Model K microscope

6 1 The Microscopemaker of the microscope. It is not easy to learn about themicroscope. But it will help you to learn if you have yourmicroscope beside you when you read this chapter.WHEN YOU READ THIS CHAPTER, PUT YOURMICROSCOPE THE SAME WAY ROLTND AS THEMICROSCOPE IN THE PICTURES. The person inFIGURE 6-2 is reading this chapter. and he has hisOlympus Model K microscope beside him on the bench.He has pslt it round exact!y the same way a. s’.e pictureof the microscope in FIGURE 6-1. In this way he caneasity find the places which all the arrows in FIGURE 6- 1point to.But, first of all, why do we use a microscope in amedical laboratory ? We seed a microscope becausemany of the things that we want to find in our patientsare too small to see with our eyes. We need a machinewith which we can see very small things. This is what amicroscope is: ‘micro’ means small; and scope means‘something for looking with’.How small are the things that we see with a microscope?To think about this it is best to start with the sizeof something we know. We will choose an ordinary footruler. This has been drawn in FIGURE 6-3. One side ofthis is divided by many short lines into inches, and theother side is divided into ‘centimetres’. Each of thesesides is a scale. One scale is in inches and the other incentimetres. Centimetres are called centimetres becausethere are a hundred of them in a metre-the word ‘cent’means a hundred. A metre is about three feet, but we arenot interested here in anything as long as a metre. On ourruler a centimotre is divided into ten ‘millimetres’, whichare the smallest graduations or divisions (little blacklines) you can see. They are called milli because ‘milli’means one thousand. There are a thousand millimetres inone metre.The smallest thing that we can see with our eyes isabout a fifth part of a millimetre across. But with amicroscope we can see much smaller things than this.We cannot use millimetres to measure these small things,because even millimetres are much too big. Instead weuse the >tm’. ‘m’ is short for a metre. ‘/l’ is short for theword ‘micro’, which, when used like this, means ‘a millionth’.As we have just seen, ‘micro’ can also be used tomean ‘small’. A !lrn is thus a millionth part of a metre.Because there are a thousand millimetres in a metre, aCtrn is also a thousandth part of a millimetre (a thousandtimes a thousand makes a million).One thousand !trn are thus equal to one of the m;llimetredivisions on our ruler. With a microscope we cansee things as small as one-fifth of a /lm! The idea of a /cmthis is a reai microscope 7standing on a benchthis is the microscopesh-)wn in Figure 6-l- - --, -‘e: _--_-- AL.------- - _ _..make sure that the microscope isthe same way round as it has beendrawn in the figuretFig. 6-2Finding the parts of the microscope

How a microscope works 1 6.2may not be easy, but unless you know about them it willnot be easy to tell you about the things that can be seenwith a microscope. We shall meet the ilrn in many of thechapters of this book.Do your best to remember that the red blood cell is 7+pm across and that a coccus (see Section 1.14) is aboutI ~rnt across. We should have to put a thousand bacteriaside by side to make a line as long as one millimetre!These are very useful sizes to keep in your mind.6.2 How a microscope worksA microscope is made of several lenses held together inmetal tubes. A lens is a piece of smooth round glass.There are two lenses in a pair of spectacles. A microscopeis like many spectacles held toge*&er in a specialWY-The thing we want to look at under a microscope, suchas a drop of our patient’s blood, is called the object. Theobject is put on a piece of clear glass called a slide. Theslide rests on the stage of the microscope. Light from alamp shines on to a mirror. Light next goes into a part ofthe microscope called the condenser and then throughthe slide and on to the object. Light from the object goesinto a part of the microscope called the objective. Thislight then goes up a long empty tube, through somethingcalled the eyepiece and into your eye. The objective isnear the object, and the eyepiece is near your eye. Lookat the microscope in FIGURE 6-1 and in Picture A,FIGURE 6-4. Find the lamp, the mirror, the condenser,the slide, the object, the objective, the tube, and theeyepiece. Don’t muddle up the object and the obiective.The object is the thing that we look at with a microscope.The objective is the group of lenses that does the looking.In describing the microscope we will follow the lightfrom the lamp through to the eyepiece. We will leave thelamp until later in this chapter.6.3 The mirror and the condenserWe put the object, such as a drop of blood, on a glassslide, because we want to shine a bright light on to itfrom underneath. This light is needed because the thingswe are looking at are very small. If they are not verybrightly lit, we shall not be able to see them clearly. Thelight comes from a lamp or sometimes from the sky. It ismade to shine on the object by going through severallenses in the condenser. Some ‘microscopes have a lampthis is an ordinaryfoot ruler\inches,and so on until 100Orum shows one pmthis diagram has been drawn\take one millimetre and \.divide it into 1000 equal.parts, this will be the\.vrn, there is only space to\.show about 20 pm here ‘.7, 6, 5, 4, 3, 2, ,O” ’the smallest thing thatwe can see with a microscopeis about this big, aboutI ;l/5 of a pm, acrossin many other figures you are showna red cell so that you can judge thesize of other thingsthis is a bacteriumand it is about 1 pmacrossthis is a red blood cell +and it is about 7% vrn across Plate 1Fig. 6-3 The /cm

.EYEPIECE------this knob moves1the tube up anddownTUBE 4OBJECTIVE -this knob moves theup and dowriris (open wide)/ 1prismcspare eyepieces ofdifferent strengthsare often suppliedwith a microscopemicroscopes have severalobjectives which can beturned round under thebottom of the tube‘MIRRORREVOLVINGNOSEPIECEeyepiece 7objectivethis is the knob for openingand closing the iris diaphragmIRIS DIAPHRAGM(looking up from underneath) \fixed just under the condenser so that the light can shinestraight into it. Other microscopes have a separate lamp,and the light from this lamp shines first on to the mirrorand then into the condenser. Because the lamp and themicroscope are not always in the same place the mirroris made so that it can move. By moving the mirror wecan shine light straight from the lamp into the condenser.The mirror has two sides. One side is flat (a ‘plane’mirror); the other side is rounded and hollow (empty, a‘concave’ mirror). Look at your own microscope and seeifvou can find the plane and concave sides of the mirror.You can take out the mirror by lifting it out of the hole itfits into in the base of the microscope. AS A GENERALRULE USE THE FLAT SIDE OF THE MIRROR. Ifyou use the light from the sky, use the concave side of themirror.Sometimes the object has to be very brightly lit; atother times less light is needed. The light shining on theobject can be altered in two ways. The light can be madebrighter by moving the condenser upwards until italmost touches the slide. The light can be made darker bymoving the condenser downwards further away from the&de. All microscopes have a special .knob for raisingand lowering the condenser. Find this knob for raisingthe condenser in Figure 6-6 and on your microscope.Turn itfirst one way and then the other way. The condenserwill go up and down.The other way of shining more or less light on theobjective is to open or close the ‘iris diaphragm’(pronounced ‘diafram’). Find the ‘iris diaphragm’ inPicture C, Figure 6-4, in Figure 6-6, and on yourmicroscope. The iris diaphragm is at the bottom of thecondenser. Look at the condenser from below. You willsee the iris diaphragm better.The iris diaphragm can be opened or shut by turning aspecial knob or ring on the condenser. Find this ‘knobfor opening and closing the iris diaphragm ’ in Figure 6-6and on your microscope. Move this ‘knob for openingand closing the iris diaphragm’ jirst one way then theother. Look at the condenser from underneath and watchthe iris diaphragm opening and shutting as you move thisknob. When the iris is open, a lot of light goes into thecondenser. When the iris is shut, only a little light goesinto the condenser.It is very important whether the condenser is high orlow, and whether the iris diaphragm is open or shut. Weshall say more about this later in Section 6.15. But wecan say now that the iris is usually kept wide open, unlesswe are using low-powered objectives with a very brightlamp, or looking at very faint (hard to see) objects. Theimportant thing is to raise and lower the condenser andto open ar;d shut the iris until you get exactly the lightyou want. Then you can see the object as clearly aspossible.nearly shut, little light\is getting through thisnearly open, much light ishole into the condensergetting through this bighole into the condenserFig. 6-4 How a microscope works6.4 Centering the condenserIn the Olypmus microscope shown in FIGURES 6- 1 and6-6 you can only do two things to the condenser. You

FROM ON TOPTHE CONDENSERB FROM THE SIDEknob for opening and closing the iris diaphragm’at one cornerTHIS IS WHAT MXI WOULDSEE LOOKING DOWN THE TUBEAFTERTHE MPIECEHASBEENTAKENOUTETHE CONDENSERtubeI Ithe condenser is straightunderneath the objective Aand ail the light is goinginto itlight frimmirror:the eyepieceIS CENTERED-properlythelightin the middlecenteredCentering the condenser 1 6.4can raise and lower it, and you can also open and closethe iris diaphragm. In some microscopes, especiallyolder ones, there are two centering screws on the side ofthe condenser so that you can also ‘center Ele condenser’.Find the ‘centering screws’ that have been drawn on thiskind of condenser in Pictures A and B in Figure 6-5.When we say ‘centering the condenser’ we mean moving itso that it is exactly underneath the objective. Only whenthe condenser is exactly under the objective will themicroscope work as it should. Find Picture E in Figure6-5. A correctly centered condenser has been drawn, andthe light from the condenser can be seen going straightinto the objective. In Pi&are F, FIGURE 6-5, the condenseris a long way away from the center of the objective,and very little light is going into it. The condenser of theOlympus microscope is centered in the factory where it ismade. Find the ‘centering screws’ in Figure 6-6 and onyour microscope. They can only be turned by a verysmal! screwdriver, and you will not need to alter them.Don’t try to center the condenser of the Olympus microscope.But if your microscope is not an Olympus microscopeand has knobs for centering the condenser, center itlike this.METHODCENTERING THE CONDENSER (IN SOME MICROSCOPES ONLY).FIGURE 6-5Turn to Section 6.14 and go as far as the end of step12. You will then have adjusted the light, the low powerobjective will be in position, the condenser wiil be raisedand will have focused on a slide. Find Figure 6-5 andlook at Pictures C and D.Close the iris diaphragm. Raise or lower the condenseruntil you see a sharp spot of light. You may see whathas been drawn in Picture C-a spot of light at one sideof the field of view. If you see this, turn the centeringscrews on the condenser one way and the other until thespot of light is in the middle, as shown in Picture D. Thecondenser will now be centered, and, unless you mcvethe centering screws, it will not need to be centeredagain. Check the centering of your microscope fromtime to time.FTHE CONDENSERIIS NOT CENTEREDIMicroscopes are made so that the condenser can betaken out. In the Olympus microscope the condenser isheld in place by a condenser holding screw.METHODslideREMOVING AND REPLACING THE CONDENSER ON THEOLYMPUS MICROSCOPE, FIGURE 6-6move the condenserto center itthe condenser is notunderneath the objectiveand the light is notgoing into itthis wayAI#Inot centeredRemove the mirror.Find the ‘condenser holding screw’ in Figure 6-6 andon your microscope. Unscrew the ‘condenser holdingscrew’ a few turns until it is loose. Don’t tako it out.Gently push the condenser down until it comes out ofthe ring that holds it.To put the condenser back, put it into the ring thatFig. 6-5 Centering the condenser

6 1 The Microscopef,ine adjustmentgaugeadjustmentlock , condenserknob for openingirisandPthe iris diaphragminside hereis’s is the filter holder,it swings out as shown bythe arrowholds it. Push it up as far as possible. Tighten the ‘condenserholding screw’.6.5 The filter holderAt the bottom of the condenser of the Olympus microscopeis a ring which holds a circle of blue glass. Thering is the filter holder, and the circle of blue glass is thefilter. Find the ‘@lter holder’ in Figure 6-6 and on yourmicroscope. Turn it out to the side and back again. Thefilter holder is made to swing out from underneath thecondenser, so that the filter can be put in and taken out.A blue glass filter changes the yellow light of an electriclamp into white likht: iike the light of the sun. Don’tuse a blue filter with the special lamp for the Olympusmicroscope (Picture A, FIGURE 6- 15), because this lampalready has a blue filter on top of the bulb.Fig. 6-6 Removing the condensertop of the stage. When the condenser is raised as far as itcan go it should be just under the slide as shown inPicture M in FIGURE 6-7.The condenser must not come too high, as has beendrawn in Picture N in this Figure. If the condenser doescome too high, it will move the slide, and the microscopewill not work well.M tke condenser is well adjusted,its top is just below the bottomof the slideGOODcondenserfilter Fmstop-=J=giggj,slidestage6.6 The condenser stopIt is very important that the condenser do& not comeabove the stage of the microscope. If it does, it will movethe slide and push it up out of its proper place. TheOlympus microscope has a special screw, the condenserstop, to stop the condenser coming up too high. Find thecondenser stop in Figure 6-6. Turn the condenser ashigh as it will go, and you will see the condenser stop hitthe bottom of the stage. The condenser stop of theOlympus microscope was adjusted in the factory, sodon’t alter it. If you have an old microscope, it may haveno condenser stop, or it may be badly adjusted; so becareful not to raise the condenser above the level of thethe condenser stop of the Olympus microscope isadjusted at the factory, don’t try to alter itthe condenser is bady adjustedits top is pushing up the slideFig. 6-7 The condenser stop

‘z:.! y,*. 4:f.‘> &&jg;y: -: r_ I -c, I I(~~. -.. 22i > ,-.--; ..These are from the Olympus microscope and are not spring loadedVIE???’iowPOWERLOWPOWERthese screwinto thenosepiece ofthe microscopeL 30dL 30e;.lens and 1short, lensquite big(objectfar fromeach otherlfocalllengthf1 ,this ringhelps youto recognizethe oilimmersionobjectivecondenserwith the middle two objectivesthe condenser is sometimesraised and sometimes loweredthe lens of thecondenser alwaysclose up againstTHESE ARE VIEWS OF THE OBJECTIVESFROM THE BOTTOMTHESE ARE THE FIELDS OF VIEWTHAT YOU WOULD SEE THROUGH THESE OBJECTIVES/ very low power objective,\this is the field of view that YOU ARE LOOKING AT Loil immersion objective,the cells look very small you would see through the oil A BLOOD FILM the cells look ver * largeimmersion objective, see howsmall it isFig. 6-8The objectives

.,,.,,)’ :_.I,,6 1 The Microscope6.7 The objectivesSo far we have only explained how we put our object onits glass slide and how it is brightly lit by the condenserwith light from the lamp. The parts of the microscopewhich make the object look bigger (the parts whichmagnify it) are the objective and the eyepiece. Likethe condenser, these are also groups of lenses in metaltubes.Sometimes, when we look at our object, we want tomake it look very big and sometimes not so big. We wantto change this magnification of our microscops. By magnificationwe mean the number of times bigger the objectis made to look. To help us to do this microscopes aremade with objectives of several different strengths or‘magnifying powers’. Most microscopes have threeobjectives, but the Olympus microscope has four objectives.They zre shown in FIGURE 6-8 and are listedi &OW.1. The ‘very low power objective’ which makesthings look four times bigger (x 4).2. The ‘low power objective’ which makes thingslook ten times bigger (x 10).3. The ‘high power objective’ which makes thingslook forty times bigger (x 40).4. The ‘oil immersion objective’ which makesthings look a hundred times bigger (x 100).This last objective is the most powerful of all. It iscalled the oil immersion objective, or, often, just the ‘oilimmersion’, because its tiny little Iens always looks at theobject through a drop of oil. AN the other objectives lookat the object through air. IT IS VERY IMPORTANTTO REMEMBER THIS.To find the magnification you are using multiply themagnifying power of the eyepiece by the magnifyingpower of the objective. For example, if you have a x 8eyepiece and a x4 objective, you are using a magnificationof 32. If you have a x 10 eyepiece and a x 100objective, you are using a magnification of 1,000.To make it possible to change quickly from one objectiveto the other the objectives are fixed to a metal wheelwhich turns round (revolves). This is the revolvingnosepiece. When one objective is being used the othersare out of the way. As you turn the revolving nosepiece,you will hear a click (a little sound) as each objectivecomes into position under the tube. This click is made bythe click stop. Find the revolving nosepiece in Figure 6-1and on your microscope. Turn it round. You will hear a‘click’ and feel the objective stop as it comes underneaththe end of the tube.It is very important to be able to recognize theseobjectives easily; so look at your microscope and makesure you can tell one objective from another. The oilimmersion objective of the Olympus microscope has aring round it so that you can tell it from the others moreeasily. Look at the objectives of your microscope. Youwill see that the bottom lens of the very low powerobjective is the biggest. The lens of the oil immersionobjective is very small indeed. The lenses of the otherobjectives come in between these two in size.The distance between the object and the bottom lens ofthe objective is very important. For every objective thereis o&y one distance at which the object is clearly seen or,as we say, in focus. When the object is in focus, thedistance of the objective from the object is called thefocal length of the objective.FIGURE 6-9 will help you to understand this. ThisFigure shows a high power objective, but other objectivesare the same. An object will only be seen clearly if itis where the dotted line AB is in Picture W. If the slide istoo close, as in Picture X, nothing will be seen. If it is toofar away, as in Picture Y, again nothing will be seen. If,as in Picture Z, the object is exactly where the line AB is,the object will be in focus, and you will see it clearly. Theviews you would see through the eyepiece have beendrawn at the bottom of the figure. In this figure theobject is a blood film, and blood cells have been drawn.in Picture Z the blood film is sharply (clearly) in focus,as you can see the blood cells easily.With the very low power objective the focal length isquite long-29 millimetres (mm). But the focal length ofthe oil immersion objective is very short-only 1.8 mm.The focal lengths of the other objectives are in betweenthese two.The focal length of an oil immersion objective is soshort that it will not focus on a film, if a slide is put filmsurface down on the stage of a microscope. This isbecause the slide is so thick that the objective cannot getclose enough to focus on the film. This is shown inPicture D, FIGURE 6- 18.When you come to use the objectives you will see thatan oil immersion objective looks at a very small part ofthe object. The very low power objective looks at a largepart of the object. We call the area of the object we cansee the field of view. The higher the magnification of theobjective, the smaller its field of view and the smaller thearea of the specimen that can be searched. This is wellshown in the pictures at the bottom of FIGURE 6-8. Thefield of view is always upside down and left to rightcompared with the object. This never matters, and youwill soon get used to it.We have already said that the oil immersion objective,which comes close to the object on the slide, looks at theobject through a drop of oil. This is a special kind of oilmade only for oil immersion objectives. It is calledimmersion oil, and no other kind of oil can be usedinstead. There will be no clear view unless the oil immersionobjective and the slide are joined by a drop of oil.The oil immersion objective will not work without oil.Because oil immersion objectives need this oil, the objecthas usually to be dry. If the object is wet, it has to beunderneath a glass coverslip, with a drop of oil on top ofthe coverslip. You will not see a clear view through amixture of oil and water.A coverslip is a small square of very thin glass whichis a little narrower than a slide. A slide is 25.4 mm (1inch) wide and 76 mm (3 inches) long. A coverslip is

The objectives j 6.7all these are the SAME objectiveA---,- - -Byou will only see anobject clearly when itlies on the line A-Band is exactly thisdistance from theobjective : thisdistance is called thefocal length of theobjectiveif the object is tooclose you will nottif the object is toofar away you wiil notsee anythingthe object is on theline A-B you will seeFig. 6-9this is what you would see if you lookthrough the eyepiece of the microscopeThe foca! lengthL blood cells in ablood filmusually 22 mm (seven-eights of an inch) square. Acoverslip is put on top of a wet object and makes a flatglass surface that can be looked at through :ur objective.The wet objects studied by the methods in this book arewet films of stools, urine, and sometimes blood. It isbetter to look at wet objects through a coverslip, especiallyif the high power objective is being used. ALWAYSUSE A COVERSLIP WHEN YOU LOOK AT A WETOBJECT WITH AN OIL IMMERSION OBJECTIVE.Picture B in FIGURE 6- 10 shows an oil immersion objectivelooking at a wet object under a coverslip. Becausethe oil immersion objective comes very close to thecoverslip, it may cause the coverslip to move on the slideand make the object that you are looking at move out ofsight. To keep a wet object still and to stop a wet smeardrying up, put a ring of Vaseline (a kind of grease) andparaffin wax (what candles are made of) round the edgeof the coverslip. This Vaseline is shown in Picture B.How to put it on i’s described in Section 7.23. It is noteasy to look at wet objects with an oil immersion objective,so wait until you are well practised with a microscope.In Section 10.2b you will read about the use of ‘Cellophane’coverslips for examining the stool. If they are

6 1 The MicroscopeA WET A DRY A DRYOBJECT OBJECT OBJECTas a stool smearsmearACLEAR VIEW A CLEAR VIEWFig. 6-10 Using an oil immersion objectivefilmA CLOUDYVIEWsoaked in saline first these ‘Cellophane’ squares can beused for other methods. They even work fairly well withan oil immersion objective.Picture C, FIGURE 6-10, shows an oil immersionobjective looking at a dry object such as a blood film.The jilm must be quite dry before the oil is put on. Thefilm is smeared (covered) with oil, and the objective dipsstraight into the oil.The objective and the slide must be joined by oil. InPicture D, FIGURE 6- 10, there is oil on the objective, andthere is oil on the fihn. But the oil does not join, and thereis air between the slide and the objective. The viewthrough this objective will not be clear. Make the oil onthe objective touch the oil on the slide. The oil on theobjective will join the oil on the slide, as in Picture C,and you will be able to see clearly.As you have read, an oil immersion objective looks atthe object through oil. All other objectives must look atthe object through air. You cannot put a drop of oilunder the high power (x 40) objective and expect it towork as a (x 100) oil immersion objective! You will notsee an object clearly in this way. Zf, by mistake, oil or anyother liquid gets on to the lenses of the high power, thelow power, or the very low power objectives, CLEANTHE OIL OFF. Zf you cannot see an object clearZythrough one of these objectives, it is probably becausethere is oil on the lens. The lens of an objective must beperfectly clean. Even a fingermark will spoil the view.Read how to clean an objective in Section 6.16 andFIGURE 6- 19.It is difficult to keep a high power objective clean andfree from oil because the high power objective comesvery close to the slide (its focal length is very short).When you turn the revolving nosepiece to change objectives,it is easy to make a mistake and to let the lens ofthe high power objective touch the oil. This spoils theview that can be seen through it. When this happens,unscrew the high power objective from the nosepiece andclean its lens.You will find it much easier to keep the high powerobjective free from oil if you keep the objectives in theright order in the revolving nosepiece. If you have fourobjectives, always keep them in this order: very lowpower, low power, high power, oil immersion. If you arechanging from the low power to the oil immersion objective,don’t let the high power objective pass over the oilon the slide. Turn the nosepiece the other way round sothat the high power objective does not touch the oil. If theoil on the film is thin, the high power objective wouldprobably not touch it, but it would almost certainlytouch a big drop of oil.A special plastic oil bottle is supplied with theOlympus microscope. If you have not got a special oilbottle, put some oil in a bijou bottle and dip a clean stickinto it. A drop of oil will remain on the end of the stickand can be put on to the slides. An opened out paper-clipcan also be used. Bend one end into a little loop to holdthe oil, and bend the other end into a handle.It is usually easy to make another slide, but it is noteasy to get another objective. BE CAREFUL. For thisreason some objectives are made with a spring insidethem. Then, if the end of the objective touches the slide, itDON’T PUSHTHE OBJECTIVETHROUGH THEstage / \(concienser 1c 4Fig. 6-l 1 A warning!

.i’i (. 1. ” .The eyepiece 1 6.6FIXED EYEPIECE MOVABLE EYEPIECEthis eyepiece can bepushed from side to sidemilled edge - turnthis and the eyepiecewill go up and downLEFTRIGHTgraduation marks/ Lthis is theinterpupilarydistance/Fig. 6-12A binocular eyepiecein some microscopes the lefteyepiece is the movable onesprings back and does not get broken. We say theseobjectives are spring lotied. The objectives drawn inFIGURE 6-9 are not spring loaded but those in the equipmentlist are.The objectives of the Olympus microscope have beencarefully made so as to be parfocal. This means that, ifsomething is carefully focused with one objective, it willalso be in focus when the nosepiece is turned and it islooked at with another objective. Very little furtherfocusing will be needed. With other microscopes whichare not parfocal you may have to alter the focusing quitea lot when you turn from one objective to another.6.8 The eyepieceWe have seen that the objectives of the microscope arefixed to the revolving nosepiece. They are at the end of anempty tube called the tube of the microscope. At the topof this tube are two more lenses in another short tube.These make the eyepiece. It is called the eyepiece becauseit is close to your eye.Just as there are objectives of different power, so thereare also eyepieces of different power. The Olympus microscopeis supplied with a single eyepiece which makes theobject look ten times bigger-a x 1 Oeyepiece. Some microscopesare supplied with two eyepieces: a x 10 and a x 5.Take the eyepiece out of your microscope. You will find iteasily comes out of the top of the tube. You will see it hastwo lenses: one lens at the top and one at the bottom.The Olympus microscope in FIGURE 6- 1 has one eyepiece:it is monocular (mono = one, ocular = eye: oneeyed).More expensive microscopes have two eyepieces:they are binocular (bin = two: two-eyed). Because itstrains (hurts) the eyes less, a binocular microscope iseasier to use, especially if it has to be used for manyhours at a time. You may have a binocular microscope.If you have a binocular microscope, it is important thatyou use it in the right way.Look at the binocular head of your microscope. You willsee that it is like the one in FIGURE 6- 12. The left eyepieceis lixed (it will not move), but the right eyepiece canbe moved in two different ways. The right eyepiece canbe moved in and out by turning it one way or the otherby its milled (rough) edge. It can also be pushed from oneside to the other side. Binocular microscopesare made to move like th.is because the eyes of anytwo people are never quite the same. People’s eyes aredifferent distances apart. It is also common for one eyeto focus a little differently from the other eye. By pushingthe right eyepiece to one side or the other the personwho uses a microscope can m.ake the distance betweenthe eyepieces the same as the distance betweenhis own eyes. This distance is called the ‘inter-pupillarydistance’ (the distance between the pupils or darkmiddle parts of the eyes: inter = between). In mostpeople it is about 65 mm, but it may be as short as 55mm or as long as 75 mm. The right eyepiece has beenmade so that it can be adjusted to the right focusfor the user’s eye. Turn the milled edge on the movableeyepiece of your microscope, and you will find that itmoves. If you have a binocular microscope, adjust itlike this.METHODADJUSTING A BINOCULAR MICROSCOPE, FIGURE S-12Focus on some object such as a blood film. Look atSection 6.14 to see how to do this.ADJUSTING THE INTER-PUPILLARY DISTANCEPush the movable eyepiece from side to side until youcan see the same object comfortably with both eyes.Read your inter-pupillary distance from the scale. Rememberit and use it to adjust your microscope after ithas been used by someone else.

6’ 1 ~The~~icro&opeADJUSTING THE FOCUSClose your right eye. Look through the fixed eyepiecewith your left eye and bring the object into the sharpestpossible focus with the fine adjustment. Then close yourleft eye and look through the movable eyepiece withyour’ right eye. Don’t alter the coarse or fine adjrrstmexlts,but bring’ the object into sharp focus by turningthe movable eyepiece. Open both eyes. The objectshould be sharply in focus for both eyes. Always adjustthe focus of the movable eyepiece after you haveadjusted the inter-pupillary distance. Do this becausethe right adjustment of the movable eyepiece dependsin part upon the inter-pupillary distance.A well-adjusted binocular microscope will be muchmore comfortable to work with than one which has notbeen well adjusted.6.9 The tube, the coarse and fine adjustmentsThe microscope drawn in Picture A, FIGURE 6-4, has astraight empty tube. But the tube of a modem microscopeis always bent to bring the eyepiece to a betterplace to look through. A zGrc?scope of this kind hasbeen drawn in Picture B in FIGURE 6-4. The OlympusModel K is a miscroscope of this kind. A special piece ofglass called a prism is put into the middle of the tube tobend the light. All binocular microscopes have a prism inthe tube so that the light coming from the objective canbe cut in half to go to both eyes.The tube of the Olympus microscope is made so that itcan move round in a circle. By moving the tube roundanother person can look down it without moving themicroscope.Microscopes are made so that the objective and theobject can be moved closer together or further apart. Insome microscopes. like the one in Picture A, FIGURE 6-4,the tube and its objectives move up and down. In othermicroscopes, such as the Olympus, the tube and theobjectives stay still and the object moves up and down.This means that the slide carrying the object has tomove. The stage (the part of the microscope on which theslide rests) must therefore be made to move also. Inmicroscopes of this kind the condenser moves up anddown with the stage.In most micrdscopes the moving is done by two kindsof knobs--the course adjustment and the fineadjustment. Find the coarse and j%te adjustments inFigure 6-1 and on your microscope. ‘r)he coarse adjustmentmakes big movements; the fine adjustment makesfine (little) movements. The fine adjustment is used forgetting the object sharply into focus when it has been putnearly into focus with the coarse adjustment. Sometimesthe knobs for the coarse and fine adjustment are separate.Sometimes, as in the Olympus microscope, the knobs aretogether and are said to be co-axial (sharing the sameshaft or axle).When yov are using a microscope alwuys keep yourleft hand on thejine adjustment (your right hand worksthe mechanical stage). It is one of the signs of the goodmicroscopist that he is always moving the fine adjustmentto get a clearer picture.6.10 The mechanical stageThere are often so many slides to be looked at, that timeis wasted if the same part of a slide is looked at twice. Itis useful therefore to be able to move the slide about in aregular careful way. This can be done with a mechanicalstage like the one in FIGURE 6- 13. A mechanical stagehas two knobs: one knob moves the slide along the stage;and the other knob moves the slide across the stage.wOlympusML30k// ///stage KMFig. 6-l 3 A movableslide across the stagestageSometimes it is important to search the whole of thespecimen under a coverslip for something. It might beCSF for trypanosomes (see Section 9.14) or urine forschistosomes (see Section 8.15). FIGURE 6- 14 shows youhow to do this.METHODSEARCHING THE WHOLE OF A SPECIMEN UNDER ACOVEIRSLIP, FIGURE 6-14The thick black line round the outside of Figure 6-14is a coverslip. Start looking in the top left-hand corner. Itwill really be the bottom right-hand corner, because themicroscope turns things upside down and right to left.With your microscope you will be able to see everythinginside a circle marked A. With the mechanical stagemove the slide so that the objective goes along the linewith the arrows marked 8. You will be able to search anwea of the coverslip between lines C and D.

., -.-Lights for the microscope 1 6.11if yc IU start looking at the slidehere ! this will be your field of view_--~-se-------.._‘,a. .- :/This square is a coverslipCT.. : .I. ;. :’ 1 -. .’.’ ..\I’ &.y. “; :i.:; ..I .jg:*y.‘.;.].‘.: 1,‘ : , ‘.( -‘1.:::;.;. ...G: ;,. . . . -.. .: 1.: ..~...~.~,~~~~~;E1-. . ,.L.9 a- :*. I- ‘. y_. ‘_ .,::,K’-.’*..,- ‘_, .‘, ‘_ . ., _._’_'. ...:.-, ..'I:' ~._:..,__... 1.1 :._:.:-.,'.".,:..,..:.,: .I,. -, . -:.--. ‘. H:..~‘-‘..,‘...“:‘i-.~,..1 ..__’ _..+A ::.:.. _: y., _'.': ,.+q-~'+ '__.+g'

a light like this. If a separate battery is to be used, it willhave to be charged up in some way when it runs down.This is discussed in Section 3.8.What can be done if there is no electricity of anykind? By day it may be possible to use the sky. By nighta big torch or a paraf6n lamp can be used-eitherthekind with a wick or a pressure lamp (a”Tilley lamp’) witha mantle (a mantle is the ‘cloth’ part of a pressure lampwhich gets white hot). Point the mirror at the flame ‘orthe mantle ar!d use it just as you would on an electriclamp-follow the directions in Section 6.14. Most healthcenter laboratories will have to use the sky.6.12 The Olympus Model K miixoscopeThe Olympus Model K microscope has been put into theequipment list because it is the cheapest and best microscopefor our kind of laboratory. There are some thingsabout it which are special.For example, the Olympus microscope has three littlelines called the fine adjustment gauge. You will see this inFIGURE 6-6 and in FIGURE 6-16. If you turn the knob ofthe fine adjustment you will see that the line on the rightgoes up or down. This line is on a part of the microscopewhich holds the stage, so that tte stage goes up and downwhen you turn the fine adjustment. Go on turning the fineadjustment as far as you can, first one way and then theother. You will find that the right-hand line finally stopseither opposite the top line on the left-hand side (PictureS) or against the bottom line on the left-hand side(Picture U). There is very little movement in the fineadjustment, and these lines have been drawn to help youto use it well. Befofe you start to use the microscope,make sure that the right-hand line is in the middle betweenthe two lines on the left, as shown in Picture. T.You will then be able to focus up or down with the fineadjustment as much as you need.There is something on the Olympus microscope whichyou will not find on other microscopes. This is tie presetwhen the righthand line ishere the fineadjustment isas far up as itwill gothis is part of rhe microscopethat is drawn in Figure 66,these three lines are thefine adjustment gaugeTSTART USING YOURMICROSCOPE WITHTHE FINE ADJUSTMENSET LIKE THISTI-tI;the fineadjustment isin the middleand can go upor downthis line movesup and downFig. 6- 16 Fine adjustmentgaugewhen the righthand line ishere the fineadjustment isas far down as 4Iit will goILEFT’ RIGHT

Knowing your microscope 1 6.13foeos lock. This locks (stops) the coarse adjustment butleaves the fine adjustment free to move. If you look atFIGURE 6-6 you will see that there is a locking It-rer onone side of the microscope. It is shown here pointingdown in the locked position. The dotted line shows itpointing up in the unlocked position. The preset focuslock is very useful for getting a slide into focus quickly.Use it like this.METHODUSING THE PRESET FOCUS LOCK ON THE OLYMPUS MODEL KMICROSCOPE, FIGURE 6-6Turn the fine adjustment so that the right-hand line ofthe fine adjustment gauge is in the middle of the othertW0.Start with the lever unlocked, that is with the lever upagainst the stop in the position shown by the dottedline.Take a slide, such as a blood film, and bring it sharplyinto focus with the coarse adjustment.Lock the lever by pushing it down. Lower the stagewith the slide on it. When you raise it again, it willalways stop sharply just where you locked it before.When you look at the slide, it will still be sharply infocus. If your slides are of the same thickness you willbe able to bring another slide into immediate focus inthe same way. The fine adjustment is still free, and youcan do the final focusing with this.To change the position of the lock, raise the lever. Youwill then be able to move the stage up as far as it can go.You can lock it again in any position you like.6.13 Knowing your microscopeBefore you start to use your microscope you will have toget to know it. You will find it helpful to do these things.METHODGETTING TO KNOW YOUR OLYMPUS MICROSCOPEFirst, have a good look at it. It is not the same on theright-hand side as it is on the left-hand side. Only on theleft-hand side of the Olympus microscope is there aknob to raise the condenser. The focusing lock and thefine adjustment gauge are only on the right-hand side.With the help of the pictures in this chapter find all thefollowing parts of the microscope. The most helpful pictureis put in brackets after the name of the part. Thebase (6-l I. the arm (6-l 1, the tube (6-l I. the eyepiece(6-l 1, the revolving nosepiece (6-l). the four objectives(6-l. 6-6). the stage (6-l). the mechanical stage (6-13).the coarse adjustment (6-l 1, the fine adjustment (6-l 1.the mirror (6-I 1, the condenser (6-6). the condenser stop(6-61, the condenser adjusting knob (6-6). the iris diaphragm(6-Q). the knob for opening the iris diaphragm(6-6). the filter holder (6-6). the fine adjustment gauge(6-6, S-161, the preset focus lock (6-6). On/y when youare quite sure what all these parts are should you readthe rest of this method.Take hold of the eyepiece and pull it out. You will findthat it comes out of the tube quite easily. Look down theempty tube. Put it back.Move the tube round, following the arrow in Figure6-l ; you will find that it goes right round the microscope.Turn the nosepiece round. Every time an objectivecomes into place under the tube you will feel a clickfrom the click stop.Turn the coarse adjustment as far as it will go oneway, and ther; as far as it will go the other way. Makesure the objectives are not going to hit anything as youdo this.Turn the fine adjustment as far as it will go one way,and then as far as it will go the other way. You will seethe right-hand line of the fine adjustment gauge moveup and down.Turn the condenser adjusting knob as far as it will goone way, and then as far as it will go the other way. Thecondenser will move up and down.Find the knob for the iris diaphragm and move thisone way and the other way. The iris diaphragm will ape!?and close.Unscrew the condenser holding screw. The condenserwill come loose and can be gently lowered out of thering that holds it. Take the condenser out. Put it backand screw up the condenser holding screw to hold ittight. You are shown how to do this in Section 6.4 andFigure 6-6.Find the lever of the preset focus lock. Move it backwardsand forwards. You will find, when you try to movethe coarse adjustment with the lever down, that thecoarse adjustment is locked and that the stage will notmove as far as it will when the lever is up.Move the mirror about. Take it out and put it back. Ifthe microscope has a light, take this out and fit the mirrorinto the hole in the base.DO ALL THESE THINGS WITH CARE. DO NOTBREAK OR STRAIN ANriHING.6.14 Using your microscopeOnly when you are sure what all the parts are and howthey move and work should you go on to the methodbelow. Read this next section over very carefully beforeyou begin. When you really understand the method, youcan begin to do what it says. Follow the instructionscarefully and don’t move to a new instruction until youhave completed the one you are doing.METHODb:IING THE OLYMPUS MICROSCOPE TO LOOK AT A BLOODF.LM WITH THE OIL IMMERSION LENS, FIGURE 6-17Stain a blood film by the method in Section 7.12. Putit cn the stage with the film side up.1. If you can, put the microscope in a dark corner ofthe room. You will not see well if the microscope is in a

6 1 The MicroscopeA llookina down from the roof) B C..:.-.. :.11, look down the eyepieceand focus the coarseadjustment with vour left hand .Q-&+Put the It )W 1 powerobjc ective in placemepie cethe tubey/ w2, find the rightadjustment gaugeis set like this6, raise the locking9, raise the condenser- - IO; open the irisdiaf lhragm14. make sure the ’ L13, move the mirror about untilslide is still in focus you see the bottom of theby turning the coarse tube brightly lit then put theadjustmenteyepiece backD -18, turn the oil immersionective into positionI16,19, look at the objectiveput a drop of oilon the slide15. look around the film fora good place to examinewith the oil immersionobjective20, make sure that the oil onthe objective touches theoil on the slide, add somemore oil if it does notFig. 6-l 7 Using an Olympusmicroscope21, using the fine adjustmentlower the stage until theblood film is clearly in focusplace where there is much light. If you have to use the 9. Raise the condenser as high as it will go.sky you will have to use a bright place under a window, 10. Open the iris diaphragm wide.as is shown in Figure 3-l 1. 11. Look down the eyepiece. CAREFULLY turn the2. Find a stool that brings your eyes to the sameheight as the eyepiece of the microscope with your backstraight. You will not be comfortable if you use yourmicroscope for a long time with your back bent becauseyour stool is too high.3. Put the lamp close in front.of your microscope. Putthe blood film you have just made on the stage of themicroscope.4. Rest both your forearms on the table. Use your lefthand to turn the coarse and fine adjustments.5. Use your right hand to work the mechanical stage.6. See that the locking lever of the preset focusinglock is raised (unlocked).7. Turn the fine adjustment until the right-hand line isin the middle in between the other two on the left, as inPicture T, Figure 6-l 6.8. Turn the low power (x10) objective into positionunder the tube. Make sure it has clicked into position.coarse adjustment (the large outer knob) one way andthe other with your left hand until you see a sharp clearpicture of the object. You will only see a clear picturewhen the film is exactly the right distance away fromthe objective. As you read in Section 6.7 this is calledfocusing. When the picture is clear it is said to be infocus. When your blood film is in focus you will seesomething like Picture 8 in Figure 6-8 except that thecells will look very much smaller.Even though you are looking through a monocular(one-eyed) microscope, keep both eyes open. This willsoon become easier than trying to shut the eye you arenot using. You will soon learn to ‘forget’ about what thiseye sees. Use one eye for a time and then use the othereye, so as to give the first eye a rest. Try to look atsomething far away when you first look into a microscope.Try to look ‘through the microscope, not into it’.It is very important how near the eyepiece you put your

Specimens of poor contrast 1 6.15eye. Start with your eye far from the eyepiece end bringit closer until you see clearly.12. Take out the eyepiece. Look down the emptytube.13. Make quite sure that the flat side of the mirror ison top. Niove the mirror around until the whole of thebottom of the tube (the top lens of the objective) is filledwith light. If there is light in the middle but not round theedge, lower the condenser until there is light all over thebottom of the tube. Put back the eyepiece. FROM NOWON DON7 MOVE THE LAMP OR THE MIRROR. oryouwill have to adjust them again. You may find the lighttoo bright to use the low power objective in comfort. Ifso, close the iris diaphragm a little by moving the knobwhich closes it.14. Make quite sure that the slide is still in focus byturning the coarse adjustment. Lock the preset focusinglock by pushing the lever downwards.15. Turn the knobs of the mechanical stage and lookaround the film until you find the part you want toexamine with the oil immersion objective. It will not bepossible to focus the oil immersion objective on a patchof clear glass. The best part of the film to look at will notbe the same in all slides. Only practice will tell youwhich it is.16. Turn the low power objective (X IO) out of the wayof the slide.17. Put a drop of oil in the centre of the slide justwhere the oil immersion objective will be when it isturned into position. A spot of light shining on to theslide from the condenser will show you where this is.Use only the special immersion oil described in Section8.7.18. Turn the oil immersion objective into position andmake sure it has clicked into the right place. In doingthis turn the nosepiece SO that the high power objectivedoes not pass over the oil. It may touch the drop bymistake and have to be cleaned. Look at Section 6.7 tosee how important it is to keep the lens of the highpower objective free from oil.19 and 20. Look at the microscope from the side. Hasthe drop of oil on the oil immersion objective touchedthe drop of oil on the slide? If the oil drops have notjoined up, move the fine atljustment until they touch.When they touch the oil will ‘light up’.If there is already a drop of oil on the tip of the oilimmersion objective, it will usually join up with the dropyou have just put on the slide as shown in Picture C,Figure 6-10. But there may be no oil on your objectivealready, and the drop you have put on the slide may bethin and it may spread out rapidly. Then the two drops ofoil will not join but will stay separate as shown inPicture D, Figure 6-10.21. Bring the film into focus with the fine adjustment.Turn the fine adjustment one way and then the otheruntil you see a clear sharp picture. DON’T RAISE THESLIDE TOO MUCH, OR YOU WILL PUSH IT AGAINSTTHE OIL IMMERSION OBJECTIVE AND BREAK IT.The objective may also be spoilt.If you cannot focus, look from the side again and bringthe slide close to the objective. Then look through theeyepiece and gently lower the slide with the fine adjustment.If you do this you will not spoil the objective orthe slide. If you still cannot focus with the oil immersionobjective, go back to instruction 14 and try again. Is thefilm on top of the slide?As soon as you have focused the slide, lock the focusinglock.When looking at another slide, look at it first with thelow power objective, bringing the stage up sharp againstthe focusing lock. Focus it with the fine adjustment andthen use the oil immersion objective, following the instructionsfrom 15 onwards.If you use the lamp which fits into the microscope,lighting will be easier, and you will not have to adjust themirror.All these instructions may look difficult, but they arenot really so hard as they look. Three things are veryimportant.(i) Before using the oil immersion objective, alwaysfirst search for a good part of the specimen with thelow power objective.(ii) Until you have had a lot of practice, always lookfrom the side when you bring the slide and the oilimmersion objective together.(iii) Be careful about the light-if it is not as brightas you want, take out the eyepiece and move themirror until the bottom of the tube is flooded withlight.6.15 Specimens of poor contrastSo far we have only thought about one kind of object-astained slide. This is a common kind of object to look atwith a microscope. But there is another kind of objectwhich needs to be looked at differently. This is theunstained specimen with poor contrast. By ‘poor contrast’we mean that there is little difference as far as lightis concerned between the object and the liquid in which itis. In other words, the object is pale and watery and noteasy to see. Examples are cells in an unstained specimenof CSF, cells in the urine, and the smaller parasites in asaline smear of the stool. Such objects need to be lookedat differently with a microscope. Follow these instructions.METHODLOOKING AT SPECIMENS OF POOR CONTRASTFollow exactly the same steps as in the method aboveexcept that you WILL NEED MUCH LESS LIGHT. Thebest way to do this is to make the light less bright. Oneway of doing this is to move the lamp further away.Another and not so good way is to lower the condenser.With higher powered objectives, especially the oilimmersion objective, you may have to close the irisdiaphragm until you get just the light you want.

01 L ON THE LENSDUST ON THE UPPER LENSA FAULTYTHE FIELD OF VIEWTHROUGH THE LOWERPOWER OBJECTIVESIS NOT AS CLEARAS IT SHOULD BE.7A40Jthe lenses inthe objectiveare spoiltoil-THE SLIDEUPSIDE DO1THERE IS AN AIR THERE IS DIRT ONBUBBLE IN THE OILTHEOBJESIVEESEDNOTHING CAN BESEEN THROUGHTHE OILIMMERSIONOBJECTIVES ORTHE IMAGE IS NOAS CLEAR AS ITSHOULD BEabject such as3 blooc(filmi-the objectivecannot focuson this filmit is too faraway/\r THE COVERSLIP OR THE THE OIL ISOBJECT ARE TOO THICK TOO STICKYle of airdirt on theTHERE ARE DARKSHADOWS IN THEFIELD OF VIEWoileyepiece isturnedEYEPIECEupper lensTHE MIRROR ISBADLY, ADJYSTED‘slideTHE CONDENSERISTHE IRISlower lensTHE LIGHT IS POOROR THE FIELDCOMPLETELY DARKirisis closedI.---Fig. 6-l 8 Troubleswith microscopes

Troubles with microscopes 1 6.166.16 Troubles with microscopesSeveral things can go wrong with microscopes, espe-.’cially when they are old. These instructions will help youto put some of your troubles right. They are all shown inFI,~URE 6- 18.(i) THE FIELD OF VIEW THROUGH THE LOWOR THE HIGH POWER OBJECTIVE IS NOT ASCLEAR AS IT SHOULD BE.There are several causes for this.A. Is there oil on the lens?There are several ways in which you can clean an objectiveor an eyepiece. The best way to remove dust is tobrush it off with a small soft, very clean paint brush.There must be no grease (fat or oil) on the brush; sowash it in a very little ether first. If you cannot get thelens clean with a brush, use lens tissue as describedbelow. If you have no lens tissue, use a clean, soft clothwhich has been very well washed. Breathe on a lensbefore you wipe it. Little drops of very clean water willform on the lens from the water in your breath. If youcannot clean a lens in any of these ways, use lens tissueor a clean, soft cloth like this.METHODCLEANING THE SURFACE LENS OF AN OBJECTIVE, FIGURE 9-19Unscrew the objective from the nosepiece of yourmicroscope.1. Hold it up to the light. Let light shine on the lensnot through it. If it is clean and shiny, put it back in themicroscope: a dirty lens is not the cause of the trouble. Ifthe lens is oily or dirty, do the following things.2. Take a 5-cm square of lens tissue and hold it betweenthe thumb and forefirrger of your left hand. Lenstissue is best kept in booklets as described in Section2.2, ML 29.Put two or three drops of ether or xylol on a corner ofthe tissue over your left middle finger.3. Gently touch the objective on the tissue where it iswet with xylol under your middle finger.4. Touch the lens on a dry patch of tissue against oneof your other fingers. Do this immediately before thexylol has time to dry off the surface of the lens. Whendrying an objective like this. always touch it on a freshdry bit of tissue.Hold the objective up to the light again, as in instruction1, to see if it is clean. If it is not clean, start again. Assoon as the objective is clean, put it back in the microscope.Throw away the lens tissue, and don’t use it again.This instruction is only for objectives which do notdip into oil and which must have dry clean lenses. Theseare the x4, x 10, and x40 objectives. Clean oil does notmatter on the lens of an oil immersion objective, but dirton the lens will spoil the view through it.B. Is there a layer of dust on the upper surface ofthe objective?To find this out, unscrew the objective, hold it up to thelight, and look down it from the top. Dust may be seen asin Picture B. Take away the dust with a small paint brushor a bit of cotton wool on the end of a stick. It isto prevent dust gathering here that no microscopeshould ever be left standing without an eyepiece or anobjective.C. Will the microscope work if it is used withanother objective?If the microscope works well with another objective, andnothing that has been said makes any difference, then thefirst objective is spoilt and must be sent back to themakers. A faulty (broken or spoilt) objective has beendrawn in Picture C.(ii) NOTHING CAN BE SEEN THROUGH THEOIL IMMERSION OBJECTIVE, OR THE IMAGE(PICTURE) IS NOT AS CLEAR AS IT SHOULD BE.There are many causes for this. Here are some of themore important ones.D. Has the microscope slide been put on the stagethe wrong way up?If the slide is upside down you will never focus clearlywith the oil immersion objective because the thickness ofthe slide may be greater than the focal length of theobjective. This is drawn in Picture D. Make quite surethat you put the slide into the microscope with the filmon top next time.E. Is there a bubble of air or a bit of dirt in the oilunderneath the objective?This is a common cause of trouble, and the bubble cansometimes be seen moving. Move the oil immersionobjective quickly from side to side. The bubble will soonslip out of the way.F. Is the lens of the oil immersion objective dirty?Take the objective out and look at it against the light asin FIGURE 6- 19. Clean it. If the microscope is an old oneand the lens has been badly used, it may be deeplyscratched and pitted (there may be holes in it). A newobjective may be needed.G. Is the coverslip or the fluid object (a stoolsmear. for instance) underneath it so thick thatthe objective cannot get near enough to theobject?Use a thinner coverslip or a thinner layer of fluid underneathit.

i:,-j&i& ,I.. .,, a.p’-.- J” ” ..I: rL.6 1’:Thci Microscopealways touch the surface of alens very gently with the tissue!!Xylol..is alsocalled xylenexylol-4FH?alet light,shine onto thelert+end not through it, hold the lens tissr- II/ between your fin! wrlook at the objectiveand thumb7-w-Lagainst the light;1,‘:\]i, wet onlv thisarea with alittlexylolhigh power objective0use a little xylol /and wipe the lensdry immediatelyafterwardslens tissue that hasbeen torn out of abookletwet area of theFig. 6- 19 Cleaningan objectiveblot the lens dry on adry corner of thetissue : look at itagainst the light to see ifit is now really cleanH. Is the immersion oil so sticky that the objectivesticks to the slide and they move up and downtogether?Use thinner immersion oil.(iii) THE LIGHT IS POOR OR THE FIELD(VIEW) ALMOST COMPLETELY DARK.Most of the causes for this are easily put right.I. Is the mirror adjusted as it should be?Focus on a s!ide with the low power objective. Take outthe eyepiece and look down the tube. Is the bottom of thetube evenly flooded with light? If not, adjust the mirroras in Section 6.11. Make sure you are using the flat sideof the mirror.J. Is the condenser close up near the slide?Perhaps you have forgotten to raise it. In Picture J thecondenser is too low.K. Is the iris diaphragm shut?Perhaps you have forgotten to open it.(iv) THERE ARE DARK SHADOWS IN THEFIELD OF VIEW, PICTURES L and M, FIGURE6-18.If these shadows move round when you turn the eyepieceround, they are due to dirt or scratches on the lenses ofthe eyepiece. If they move when you turn the upper lens,they are due to dirt on the upper surface of the top lens,as shown in Picture M. If they only move when you turn

Some ‘DOS’ and ‘Don’ts’ in microscopy 1 6.17the lower lens, they are due to dirt on this lens. Clean thedirty lens very gently with lens tissue and xylol just asyou would an objective. The eyepiece of the Olympusmicroscope cannot be taken to pieces; so don’t try. Thecommon place for dirt to collect is on top of the top lens.(v) THE FIELD OF VIEW LOOKS OVAL AND ISNOT ROUND AS IT SHOULD BE.Perhaps the objective is not properly held in place by theclick-stop? Turn the objective into its proper place underthe tube until it is held by the click-stop. Perhaps thefilter holder is not properly in place?6.17 Some ‘DOS’ and ‘Don%’ in microscopy(FIGURE 6-20)Finally, here are some practical points to remember.Some of them are shown in FIGURE 6-20.staple them between two bits of cardboard as shown inPicture Y. This will keep the tissue away from any dustthat may scratch the lenses.Use ether or xylol (xylene) for cleaning the lenses.Don’t use spirit or alcohol because you may dissolve thecement (glue) with which the lenses are stuck into theobjectives. Spirit or alcohol may spoil your objectivescompletely.Don’t clean lenses with your tie, an ordinary handkerchief,or your coat. Use o&y the special lens tissue, or ifthis is not available, use soft paper cleaning tissuesinstead.DON’T RUB THE SURFACE OF A LENSHARD-touch it with the least possible force that willremove dirt. If there is even a tiny bit of grit (sand)and you rub hard, this will make a scratch and spoilthe lens.Be careful when you take a slide or a counting cham-keep the dust off yourmicroscopedon’t let your microscopestand around without anepiece or an objectiveclean the lenses withxylol, never use spiritzuse lens tissue anFig. 6-20 Some ‘DOS’ and ‘Don’&’ in microscopycut up big sheets of lens tissueand make them into bookletslike thisDust is a great enemy of microscopes; so keep dust offyour microscope when you are not using it. Many microscopesare supplied with a plastic cover as is theOlympus microscope drawn in Picture W. If your microscopehas not got a cover, either keep it in the box inwhich it came or make it a cover of plastic sheet or cloth.Keep the tube of the microscope closed. If you leave amicroscope without an eyepiece or an objective for anylength of time, dust will get in and may cause trouble-.see Picture B, FIGURE 6- 18. If you have not got aneyepiece or an objective to put in the microscope, closeup the hole where they go with a bit of tape or cottonwool. Better still, use one of the special caps or plugs thatare made to close these holes. In Picture X, FIGURE6-20, dust has been drawn getting into a microscopethrough the holes left by an eyepiece and anobjective.Use lens tissue and keep it in booklets about 6 cmsquare. Cut up the big sheets in which it is supplied, andber from the stage cf the microscope. Don’t rub themagainst the objective-it may be scratched. Always raisethe objective before removing the slide. Don’t leaveslides or counting chambers on a microscope.Keep the objectives in the right order in the revolvingnosepiece-very low power, low power, high power, oilimmersion.Make sure that your microscope works. Zfvou cannotmend it and make it work, try very hard to get a new one.In some microscopes the coarse adjustment is so loosethat the tube falls down and lets the objective touch th?slide. Such a microscope cannot be used.When you put your microscope away always see thatthe low power objective is in place under the tube. Thiswill stop the oil immersion objective being pushedagainst the condenser by mistake.Clean the oil immersion objective with lens paperbefore you put your microscope away at the end of theday.

6 1 The Microscope ’6.18 Looking after microscopes in warm, wet countriesIn dry countries microscopes are usually quite safe. But,if the air round a microscope is warm and wet, fungi maygrow on the lenses and spoil them. Polished glass lensesbecome cloudy and you will not be able to see throughthe microscope clearly. In some very warm, wet countriesa microscope may be badly spoilt in a few weeks. Whatcan be done? The best way of looking after a microscopein warm, wet places is to put them in a thick polythenebag with a desiccant, such as silica gel. A desiccant is achemical which takes up water from damp air and makesit dry. Fungi do not grow on lenses if the air is dry, ornearly dry. Silica gel is listed as Choice 3, ML 118a inSection 13.16. When this kind of silica gel is dry, it isblue. when it is wet it goes pink. Use it like this.METHODLOOKING AFTER A MICROSCOPE IN A WARM, DAMP CLIMATEGet a thick polvthene beg (Ml. 118b). Thick bags keepout the wetet better than thin ones. Make sure it isairtight, by closing the top and squeezing it gently. Closeany holes there may be. You may be able to close themwith adhesive tape (sticking plaster).If your silica gel is pink (wet). heat it in a clean panover a stove, mixing it all the time. It will soon go blue.Do not overheat it.Put plenty of dry blue silica gel (not less than 250 g)into a shallow dish or tin (ML 11 &I. Put this dish intothe bottom of the bag. Put the microscope into the bagon top of the diih of silica gel.Fold over the top of the bag several times, and keep itclosed with a clip such as a large paper clip. Test thatthe bag is airtight by squeezing it gently.Keep your micrvscope in this bag whenever it is notbeing used.As soon as the silica gel goes pink, beet it up until itgoes blue again.Your microscope will be safer if you always keep itslenses clean. If you have not got a bag and silica gel itwill be safer out on a bench than shut in its box, or underits plastic cover. It will also be safer near a fan. Anotherway of looking after a microscope is to keep it warm in acupboard over an electric light bulb that is always on.There should be holes in the cupboard so that the air canmove freely. Yet another way is to keep a microscopeunder a metal cover or hood which is heated by anelectric light bulb that is kept burning, But none of theseways seem to be as good as keeping a microscope in apolythene bag with silica gel as a desiccant--but onlyprovided it is kept dty and blue.QUESTIONS1. What is a Corn? How wide is a red blood cell?2. Make a list of all the parts of a microscope throughwhich light shines as it goes from a lamp to youreyes.3. What is the condenser of a microscope? Whatadjustments can youmake to it? Why are they made?4. What differences are there between a high power(x 40) objective and an oil immersion (x 100) objective?What differences are there in the way these objectives areused?5. How would you clean the surface of a low power(x 10) objective that had got oil on it?6. Why is a binocular microscope so useful? Whatadjustments can be made to the head of a binocularmicroscope?7. How is the preset focus lock of the Olympus microscopeused?8. What different adjustments to a microscope areneeded when looking at an unstained specimen of CSF,as compared with a stained blood film?9. What might be wrong if the field of view through anoil immersion objective is cloudy?10. How can you light your microscope if you haveno mains electricity?

.,:‘=--L&,??6;,* >.: -- ,I i .~:. -,‘.’ ,Ir ^,.;,7 1 BloodANAEMIA7.1 HaemoglobinThis is the red substance in the blood cells that makesblood red. When people have less haemoglobin in theirblood than they should have, their blood is pale and theyare said to be anaemic. Anaemia is very common, so it is.useful to be able to measure it. We can see if a patient is’anaemic in two ways. We can measure how muchhaemoglobin there is in his blood and we can also measurewhat is called the haematoerit. Let us first thinkabout ways of measuring the haemoglobin.Haemoglobin can be measured in two ways. We canmeasure it as oxyhaemoglobm, which is haemoglobinmade bright red by the oxygen in the air. We can alsomeasure it more accurately, but with slightly greaterdifficulty, when we change it into a substance calledcyaamethaemoglobim. The cyanmethaemoglobin methodis described with the EEL calorimeter and the standardfor it in Sections 5.19 and 5.20.Three methods of measuring oxyhaemoglobin aredescribed here. They have all been partly explained inChapter Five, where the measuring instruments weredescribed. Zf you are still in doubt when you have readthis section, look back to Chapter Five. All three methodsstart in the same way. 0.05 ml (jr, ml) of blood is addedto 10 ml of haemoglobin diluting fluid as described inSection 3.3 1. Blood can be taken from an ear prick orfrom a finger prick-these are capillary specimens.Blood can also be taken from a vein-a venous specimen(see Section 4.7).Take blood with the special blood pipette (ML 32).This has three marks on it, one at 0.02 ml, one at 0.05ml, and one at 0.1 ml. For a!1 the haemoglobm methodsO-05 ml of blood is added to 10 ml of diluting fluid.Sometimes it is dimcult to get 0.05 ml of blood out of apatient’s ear or finger. and it may be convenient to addO-02 ml of bltiod to 4 ml of diluting fluid.The blood pipette must be clean and should also bedry before you use it. The best way to clean a bloodpipette is to use an inflammable and volatile liquid calledaeetone. If you have acetone, use it like this. Put someacetone in a small bottle or tube, and fill another tubewith water. First clean the pipette by sucking the waterup and down. Then, when the pipette is clean, suck upsome acetone. Last of all suck air through with a filterpump until the pipette is dry.If you have no acetone, yet will have to use yourpipette wet, which will give you a less accurate answer.When you use a venous blood specimen it is possible toovercome some of the inaccuracy of a wet pipette byfilling it once or twice with the blood specimen. In thisway the blood can be used to remove the last drop ofwater in the pipette before it is filled. In the same wayblood can be washed out of the pipette into the dilutingfluid.MlETHDDMEASURING HAEMOGLOBIN BY THE OXYHAEMOGLOBINMETHODA. USING THE LOVIBOND COMPARATOR, FIGURE 7-l1. Fill a Lovibond tube to exactly the 1 O-ml mark withhaemoglobin diluting fluid-no other tube will do.Making this fluid is described in Section 3.31. If you findit difficult to get the meniscus of the fluid to exactly thelo-ml mark, put in a little too much and then use aPasteur pipette to remove the extra fluid.Haemoglobin diluting fluid does not keep well, sodon’t keep it more than a week or so and don’t make upmore than 100 ml at a time.2. Using one of the glass chips described in Section4.7. and a piece of cotton wool soaked in spirit, takeblood from a patient’s finger (3). or from his ear (4). Youcan also use a venous specimen (5). but remember tomix the bottle before you do so, because the red cellsmay have sunk to the bottom. When you take a capillaryspecimen remember to wipe away the first drop, and tofill your pipette from the next drops. The first drop maynot be a true sample of the patient’s blood.6. Put a rubber tube and mouthpiece on to the drypipette. Dip the end of the pipette into the blood specimen.Hold’the pipette sloping slightly downwards asin Picture 7. If the tip of the pipette is slightly higherthan the other end, blood will go in by itself, and there isno need to suck. Don’t hold the pipette vertical (upright)as in Picture 6.9. Don’t get any bubbles into the blood in the pipette.

- 2aglasschip 3/ 1 mouthpiece,-IIL--g.$ ;@$10 $&!:M ::: .::. g$?y ;:.:.::, ...?:DON’Thold thethis is the older kind ofround Lovibond ” tnha .-.,-, thn. ..”“ewer I LUIIUYlld ^.,%..-... cells arecn..~..~ yucan 2DON’T get air bubbles into the bloodthis is a venousblood specimen ina Sijou bottlemeniscus\mix the blood beforefilling your pipette9 /no!Y-ILthe pipslightlydownwards asyou fill it(/agauze orcotton wool10ML32b&: \the outside ofthe pipette is cleanfill the pipette to justabove the 0.55 ml mark :hold the pipette level :let the mouthpiece fallfrom your lips : touchthe end of the pipetteon a bit of gauze a fewtimes until the meniscusfalls to exactly 0.05 mlWIPE THE OUTSIDE OF THEPIPETTE CLEAN OF BLOODblow the blood intothe diluting fluid :draw the fluid up tojust above the 0.05 mlmark : blow it backinto the tube andblow the pipette drythis is the Lovibonddisc number 5/37xfor oxyhaemoglobinfill this tube with clean waterL-iWITH THIS SCALE YOUCAN CHANGE HAEMOGLOBIN .-.. - ----IN GRAMS % INTHE HPCDANE-thetestsolutionML 13ML 2lathis is theedge of the discanswer hole : read3-40the haemoglobina-’here in grams 1per 100 ml of bloodFig. 7-lThe haemoglobin

The haematocrit 7.2If you do. you will not measure the right volume ofblood, and your answer will not be accurate. If you getbubbles into the blood, blow them out and start again.Fill the pipette to just a little beyond the 0.05ml mark.Then carefully clean the outside of the pipette. Ifyou leave any blood on the outside, it will be washedoff into the Lovibond tube, and the answer will be toohigh.10. Then, still keeping the pipette flat, touch its endwith a piece of gauze or cotton wool. This will suck alittle blood out of the pipette, and the meniscus caneasily be brought to the 0.05ml line exactly. Make surethat the outside oFthe pipette is clean.11. Blow the blood into the fluid in the Lovibondtube. Suck some fluid back into the pipette to justabove the 0.05ml mark, but no further. Blow it outagain two or three times, because the pipette is made tobe used in this way. This will wash out the bloodsticking to the inside of the pipette. It will make surethat all the blood is in diluting fluid and will clean thepipette at the same time. This is the haemoglobin testsolution.Hold your thumb over the tube and mix it gently.12. Make sure that the Lovibond haemoblobin discmarked 537x is in the comparator, and that the whitecircles with the answers on them are looking towardsyou.13. Hold the comparator up to the daylight and turnthe disc until the view through the holes looks the same.Read the answer through the answer hole. This will tellyou the number of grams of haemoglobin the patienthas in each 100 ml of his blood. For short we write this‘grams per cent’ es ‘g %‘.14. This picture has a scale on it with which you canchange g % into ‘per cent Haldane’. Thus 6 g % is almostexactly 40% Haldane. The scales do not match exactly,and you will have to look carefully when changing oneinto the other. Thus 2 g % is a little less than 15%Haldane. say 14% Haldane.More is said about the Lovibond comparator inSection 5.10. Follow these instructions carefully.B. USING THE GREY WEDGE PHOTOMETERUsing a lo-ml straight pipette (ML 35 c) put 10 ml ofhaemoglobin diluting fluid into a universal container (ML14b). Add 0.05 ml of blood as described above. Mix welland pour the mixture into one of the cells of the Greywedge photometer. Wipe the outside of the eel! clean.Put it into the right-hand place in the cell compartmentof the photometer. Fill the left-hand cell with cleanwater. Make sure that you are using the green No. 2eyepiece. Match the two halves of the visual field. Readthe scale reading on the wheel. Either report this as ‘percent’ or change it into g % using the scale in Picture 14,Figure 7-l.More is said about the Grey wedge photometer inSection 5.11. Read these instructions carefully. anddon’t forget to check with the grey glass standard.C. USING THE EEL COLORIMETERDilute 0.05 ml of blood in 10 ml of haemoglobin dilutingfluid, exactly as described in part B above. Measurethe haemoglobin as described in Section 5.20, using theliquid grey standard, the green llford 625 filter, and thecalibration graph shown in Figure 5-10.There may be a hundred women or more at anantenatal clinic who all need their haemoglobin measuredon the same morning. What is the quickest and best wayof measuring so many haemoglobins? A good way is toget many universal containers and to fill them with 10 mlof diluting fluid before the clinic starts. Hand eachmother in the queue a bottle of diluting fluid for herhaemoglobin and then go down the queue measuringtheir haemoglobins using a Lovibond comparator, or aGrey wedge photometer (this can be used with daylight).Try to get someone else to help you, and write a mother’shaemoglobin on her card immediately you have taken it.Have some bottles with 4 ml of diluting fluid ready, sothat if you cannot get the full O-05 ml of blood, you canput 0.02 ml of blood straight into 4 ml of diluting fluid.7.2 The haematocritThis is another way of testing for anaemia. If you havethe right instrument it is easier and more accurate thanmeasuring the haemoglobin, and can often be usedinstead of it.In Section 1.17 you read how, when blood is preventedfrom clotting and left to stand for some hours, thered cells fall to the bottom and leave clear plasma on top.In Section 1.5 you also read how red cells and plasmacan be separated much more quickly by centrifuging. Ifblood is centrifuged very fast the red cells are soonpacked (pushed) tightly together at the bottom of thetube. When you look at normal blood after centrifuging*you will see that the packed red cells (they are usuallycalled packed cells) take up slightly less space than theplasma. In every 100 ml of blood from a healthy personthere will be about 45 ml of packed cells and about 55 mlof plasma. Another way of saying this is to say that thevolume of the packed cells is 450/o of the volume of theblood. The volume of packed cells is called the packedcell volume or PCV. The haematocrit is the same as thePCV and is the word we will use here. The normalhaematocrit is therefore about 45%.If we take some anaemic blood and centrifuge it, wewill find that the haematocrit is less than normal, perhapsonly 30% or even only 10%. We can therefore measureanaemia by measuring the haematocrit as well as bymeasuring the haemoglobin. An anaemic patient has lessthan the normal amount of haemoglobin. He usually alsohas a haematocrit which is less than normal.We shall describe two instruments for measuring thehaematocrit. One is the MSE Minor Centrifuge and thepieces of equipment that go with it. This centrifuge isuseful because it can be used as an ordinary centrifugefor urine, as well as for the microhaematocrit. The other

fill the tube until itis about as full as this1 tap the end of the tube so thatthe end of the tube is left emptythis is the lid thatthis is the microhaematocrithead of the MSE centrifugehave no tubeinside theminside the centrifugeafter centrifugingthe lid has beenclosed and then IV.,,sIl&--plasmaH ‘nmuchplasmathe ~~of the cursorthis is the haematocrit ---from a very anaemic patient,the top of the plasma it is only about 20%is on the sloping lineI very smallll Innntl7 ‘L”$J”’ A+ “Ipackedcellswith a line on iof the colum./the segment+-- scale7iire segmentcan move up anddown in the clamp‘the bottom ofuthe packed cellsis on the base line;-- T-------7--read the haematocritfrom this scaleFig. 7-2The haematocrit

z -.The haematocrit 1 7.2instrument is the Taylor-Eaves microhaematocrit, whichcan only be used for this method, but which is muchcheaper:The MSE MinorcentritkgeBesides the centrifuge itself (ML 122), you will need thespecial head for it (ML 122b), heparinized capillarytubes (ML 122f), and the MSE haematocrit reader(ML 122~). The heparinized capillary tubes are thinpieces of glass tube which have a small quantity ofheparin inside them. Heparin is an anticoagulant, and,like sequestrene, it stops blood from clotting. The microhaematocritreader is shown in Pictures 14 and 16.METHODTHE YSE MICROHAEMATOCRIT. FIGURE 7-21. Take a heparinized capillary tube and pti its endinto a drop of blood taken either from the ear (21 or fromthe finger. The tube can also be filled from a venousblood specimen taken into a sequestrene bottle (3). Thetube may fill more easily if you hold it on its side asshown in this picture.4. The tube should be about three-quarters full. Tap itgently on one end so that the column of blood comesclear of either end, and is closer to one end than theother.5. Hold the more empty end of the tube for a moment_ in a flame so that its end seals up. Don’t hold it in theflame too long or the tube will bend. The end of the tubewill seal, and as soon as it does so, the blood will movealong the tube a little. This is a sign that the tube issealed.6 and 7. Put the sealed end of the capillary tube intothe carrier segment. Write the patient’s name in greasepencil on the side of the segment. The tube lies in adiih or groove in the top of the segment and in a hole atits bottom.6 and 9. Put the carrier segment with its tube insideinto the head of the centrifuge. Make sure that the headis full of carrier segments, with or without blood in them.Put on the lid. IF THERE ARE LESS THAN TWEL’dESEGMENTS INSIDE THE HEAD BEFORE IT ISSTARTED, THE CENTRIFUGE WILL NOT RUNSMOOTHLY AND IT WILL BE SPOILED.IO. Run the centrifuge as fast as it will go for tenminutes.Fooinote. The designs of both centrifuges were changed by the makersas this book was printing. The equipment listed in Section 13.7 hasbeen altered, but it has not been possible to change Figure 7-2. Carriersegments are now no longer used in the MSE Minor centrifuge. Instead,the filled capillary tubes are placed in slots in the rotor, which is closedby a cover. Also, a better way of sealing the capillary tubes can be usedwith both instruments. Instead of sealing the tubes in a flame, their endscan be pressed into a thin (5 mm) layer of a plasticine-like sealingcompound held in a special trav (ML 122e). This fills the ends of thetubes with sealing compound id‘ seals them. These sealed ends thenpress against a plastic sealing strip or gasket at the edge of the rotor.11. Take the carrier segments out of the centrifuge,You will see that there is a column (line) of packed redcells at the bottom of the tube, and at the top of the tubethere is a column of clear plasma. In normal patientsthere will be nearly as much red cells as plasma, as inPicture 12. In anaemic patients there will be muchplasma and only a short column of red cells, as inPicture 13. These pictures are only to show you whatyou might find-don’t take the capillary tubes out of thecarrier segments.14. Put the carrier segment with its capillary tubesinside into the clamp (holder) of the microhaematocritreader. Move the carrier segment up and down until thebottom of the red cell column is opposite the base lineof the reader. Move the clamp along the reader until thetop of the column of plasma is opposite the sloping lineof the reader.16. Without moving the clamp, move the cursor sothat its centre line is opposite the top of the column ofred cells. The cursor in this reader is only a strip of clearplastic with a line on it. Look along the centre line of thecursor, and read off the haemoglobin from the scale. Becareful not to move the clamp while doing this. Picture15 shows this in more detail. You can see the base lineopposite the top of the column of plasma. The centreline of the cursor is opposite the top of the column ofpacked cells.The Bickerton-Eaves microhaematocritThis is a simple instrument made of an electric motor towhich is fitted a plastic head or rotor. It is housed in ametal box with a lid and has a time switch which turnsoff the motor after any time up to 15 minutes. Thisinstrument will take up to 24 capillary tubes and is listedas ML 123a in Section 13.17. There is also a 12-voltmodel which can be run from a car battery.METHODTHE BICKERTON-EAVES MICROHAEMATOCRIT, FIGURE 7-31. Fill and seal heparinized capillary tubes exactly asdescribed above.2. Lift the cover of the case. Place your sealed capillarytubes in the numbered slots in the rotor.3. Close the lid and turn the time switch to 15 minutes.The machine will run and then turn itself off at theend of this time.4. Raise the lid. Read each tube by sliding it alongthe chart that is supplied with this machine and shownin Figure 7-3. If you have no chart, use the one at the endof the book which you can tear out. Put the bottom ofthe column of blood on the line marked 0. Slide the tubealong the chart until the top of the column of plasmajust touches the sloping line marked 100%. Read whichof the other sloping lines the top of the column ofpacked cells touches. This is the haematocrit reading.Never run the machine with the lid open.

,slots forcapillarytubes__-rK I-cover-threedischolesrotorthreescrewheadshaemoglobin and have a normal pink colour on a stainedfilm. These are the normochromic, or ncrrmal-colouredanaemias, in which the haemoglobin and the haematocrithave fallen in proportion to one another. But, someanaemic patients lack the iron they need to make thehaemoglobm to fill their red cells. The haemoglobin inthese patients thus falls more than the haematocrit andtheir red cells are pale and partly empty of haemoglobin.Red cells which are pale and partly lacking haemoglobinin this way are said to be hypochromic. As you will readin Section /‘.19, it is possible to see if red cells arehypochromlc by staining a blood film and looking atthem under a microscope. But this is not always easy,especially if hypochromia is mild, and a better way tomeasure it is to combine the haematocrit and the haemoglobintogether and work out a special measure of howmuch haemoglobin there is in red cells called theMCHC.The MCHC stands for the Mean (average)Corpuscular (a red corpuscle is a red cell) HaemoglobinConcentration and is worked out like this:Haemoglobin in g % x loo = MCHC o/0Haematocrit %The MCHC of a normal person is therefore:CHART FOR THE HAEMATOCRIT/nput the capillarytube on top ofthe charthiom of red cellsH. Bickerton Ltd. Beehave Works. Wslwn. England. level with thebottom lineFig. 7-3 A cheaper instrument for the haematocrit7.3 Working out the MCHC from the haemoglobinand the haematocrit/ -lluoWe have just seen that there are two ways of finding if apatient is anaemic-we can measure his haemoglobin,and we can measure his haematocrit. These methods areboth measures of anaemia, but they measure differentthings. When we measure the haemoglobin, we measurehow much of the red substance called haemoglobin thereis in 100 ml of blood. When we measure the haematocrit,we measure the percentage of the blood that is taken upby red cells. Different kinds of anaemia alter the haemoglobinand the haematocrit in different ways. Thus, inmany kinds of anaemia there are fewer red cells thanthere should be, but these few red cells are well filled with+x lOO= 33%Any MCHC between 32 and 36% is normal. Normal redcells are already as full of hnnemoglobin as they can be,and if you get a figure of over 360/o, one of your methodsmust have been wrong. Either the haematocrit is too low.or the haemoglobin is too high. Any figure below 32%means that the patient’s blood is hypochromic. To helpyou find the MCHC a special diagram called a nomogramhas been drawn in FIGURE 7-4. To USA this nomogramfirst find the patient’s haemoglobin on the top scale.Then fmd his haematocrit on the bottom scale. Join themup with a ruler. Find where the ruler crosses the middlescale-this is the patient’s MCHC. The dotted line in thefigure gives you an example. The patient’s haemoglobinwas 8.1 g % and his haematocrit 34%. The dotted linecrosses the MCHC scale at about 24%, so his MCHCwas 24%. There is a special nomogram for you to tearout at the end of the book.It is important to be able to measure hypochromia,because it is common, and it means lack of haemoglobinin the red cells due to lack of iron. Iron medicines arecheap and hypochromic anaemias are usually easy totreat.Hypochromia can be measul;ed in another way. This iswith a special chart. The next section describes such achart for children.7.4 An anaemia chart for the ‘Under Fives Clinic’An under fives clinic is a special clinic to which mothersbring their children from the time they are born untilthey are 5 years old. Many of the children who come to

An anaemia chart for the ‘Under Fives Clinic’ i 7.4Haemoglobin 9%Lvery lowhaemoglobinvery low MCHCincreasing anaemia.M.C.H.C.%I/normalthe dotted line is an example of how touse this nomogram : the patient’s haemoglobinwas 8.1 g % and his haematocrit 34% : a ruleriHaematocrit %:c80 70 60 50 40:’ 3020 15 1011l1111l11111111,lIII 111, I, I I I I I l11r1.1.1.1, I, I, I , I ,I I I ,~&y.&&~~;~ /i ,................ :.::” .,:...:, increasingly low haematocrit very low1 .*.‘.-A%.. . . .. . .-. . . . . . 1haematocriti~~~~~~~~~~ ,. I InormalFig. 7-4 A nomogram for the MCHCthese clinics are anaemic. The cause of their anaemia hasto be diagnosed by the methods in this book, after whichthese children must be treated. Because treatment maytake some weeks, or even months, it is useful to have achart on which a child’s haemoglobin or haematocrit canbe recorded. The person who is treating the child canthen see if his anaemia is improving as it should. FIGURE7-5 shows an anaemia chart which a mother can keepwith the weight chart that she brings with her child tothese clinics. You will see that the inside is divided intothree parts-one part for each of the first 3 years of achild’s life. The parts for his fourth and fifth years are onthe outside. Along the bottom of the chart are five rowsof twelve boxes. There is a box for each month of achild’s life. The first box of each row has a thick blackline around it and is for the month of his birth. The otherboxes are for the months after that. You will see that thechart in the figure has been filled in for a child born inMarch 197 1.These charts have the haemoglobin written down theleft-hand side, and the haematocrit written down theright-hand side. They can thus be used with either ofthese methods, the haemoglobin being recorded with aspot, and the haematocrit with a cross. But it is evenmore useful to do both these methods on an anaemicchild. If the cross for the haemoglobin is in about thesame place as the dot for the haematocrit, his anaemia isnGrIIIGChrOtniC, and he does not need iron treatment. But,if the cross for the haemoglobin comes below the dot forthe haematocrit, his red cells are poorly filled withhaemoglobin, he has a hypochromic anaemia, and heneeds iron treatment. This is what was wrong with thechild John in the figure who had quite a severe hypochromicanaemia due to hookworm infection that wastreated with iron. When he was given iron his redceils filled with haemoglobin, became normochromic,and his anaemia improved. You can see this happeningon the chart which shows the crosses for thehaemoglobin climbing up and joining the dots for thehaematocrit.We have seen that when the crosses fcr the haemoglobinand the dots for the haematocrit L ’ close togetherthe cells are nOrmGChrGmiC. We have also seen that whenthe haemoglobin is below the haematocrit, the cells arepoorly filled with haemogiobin and are hypochromic.However, the haemoglobin can never lie much above thehaematocrit, because healthy normochromic red cellsalready contain as much haemoglobin as they can, andcannot contain any more. If, therefore, the haemoglobincomes much above the haematocrit on the chart, somethingis wrong, and either the haematocrit is too low, orthe haemoglobin is too high. When this happens checkyour methods.Children have lower haemoglobins and haematocritsthan adults. Both the haemoglobin and the haematocritare high at birth, but they fall quickly at first, and thenmore slowly after that, so that by the time a child is ayear old his haemoglobin is only about 10 go/o and hishaematocrit about 33%. From the age of one yearonwards they slowly rise again. The normal values for

IILa_P ,‘A,,/. ‘-‘.Haem&lobioHaematocrit3-4 vears 4-5 vears12111c6Under fives clinicANAEMIACHARTCHILD’S NAME & ADD=TO COMPLETET==I & 135ICHARTFill in the first box (with the thick border)with the month of the child’s birthday.Repeat every year and then fill in all theother months.Example: A child born in April , 1970Test the child for anaemia every monthby measuring his haemoglobin and/orhis haematocrit. In a busy clinic onlyone of these tests need be made.Make a cross in that month’s columnfor the child’s haematocrit. Make aspot for his haemoglobin. Join thespots and crosses month by month.Use the rest of the card to record thetreament given to a child. An exampleis shown of a child having iron andantimalarial treatment.Normal valuesThe average normal haemoglobinand haematocrit are shown by thedotted line on the chart. After thefirst few months of life a haemoglobinbelow 10 9% shows thata child has anaemiaNormochromic anaemiasThe haemoglobin and haematocritresults overlap.Hypochromic anaemiaThe haemoglobin result falls wellbelow the haematocritNote. An ordmary weight chart from an under fives clinic can be used. Plot haemoglobin in 9% on the kilogram scale.The corresponding haematocrit value can be found from the graph below. Thus 10 kg, a haemoglobin oflog%, and a haematocrit of 30% all use the same line.~~ -~Haematocrit ; Haemoglobin Haematocrit a Haemoglobin HaemntnrrilZwks-1 vear15l-2 years445 ’ 15 2-3years8I4 42 ’IllIi II I 1111':39 ,rne naemoglohrn and haemaihealthy child follow this line36ILnt: rlaemarocrn .._.. 1s marKea1 -“- I) the haemoglobin-and the haer&oc;itare about the same . the anaemia IS‘“‘Whnormochromic 1 1 1 ‘1’ 1 wI ‘h939 21 ; 9 37I ,8 24 I 6 24II I II I I I 8 1 Hz1 ’ VI-t-3. la1the haemoglobin is below the haematocrittheanaemia is hypochromic ! ! . _ _montlbirthn is marl15 I,1Fig. 7-5 An anaemia chart for the ‘Under-Fives Clinic’

Causes of anaemia 1 7.5both the haemoglobin and the haematocrit are shown bya dotted line on the chart.These are useful charts, both for the haemoglobinalone and especially for the haemoglobin and the haematocrittogether. But, if they are going to be used as ameasure of hypochromia, both the haemoglobin and thehaematocrit must be accurately measured. This may notbe easy in a busy clinic.7.5 Causes of anaemiaNormal men have between 14 and 18 g of haemoglobinin 100 ml of their brood and a haematocrit of between 42and 54%. Normal women have about 2 g less haemoglobin,and a haematocrit which is about 5% smallerthan men (a haemoglobm of 12 to 16 g % and a haematocritof 36 to 48%). In normal people the haemoglobinis thus about three times the haematocrit (3 x 15 = 45).14.8 g % of haemoglobin is often taken as an averagenormal and is discussed in Section 5.13. Patients whohave less haemoglobin than this are said to be anaemic.A very anaemic patient would have less than 5 g % ofhaemoglobin and a haematocrit of about 24%. A mildlymoderately anaemic patient would have ahout 8 g % ofhaemoglobin or a haematocrit of about 24%. A mildlyanaemic patient might have a haemoglobin of 10 g % anda haematocrit of 30%.What causes anaemia? A patient may become anacmicbecause too few red cells containing haemoglobin arebeing made by the body. If the body is to make enoughhaemoglobm it must have the right things to makehaemoglobin with. Some of the more important thingsthat the body needs to make haemoglobin are iron,protein, a vitamin called folic acid, and a vitamin calledI .wtamm B,* A vitamin is a food which the body musthave but only needs in small amounts to keep healthy. Ifthere are not enough of these things in the food, or ifthey are not properly absorbed (taken up) by the body,the patient will have a deficiency anaemia. A deficiencymeans a lack of something, or that something ismissing.A patient may also become anaemic because too manyblood cells containing haemoglobin are being lost bybleeding outside his body or because they are being destroyed(lysed) inside his body. Patients sometimes loseblood into their gut, as when they have hookwormswhich bite the wall of the gut and cause bleeding. Eventhough this blood is being lost into the gut, it is being lostoutside the body, and leaves the body in the stools. Bloodmay be lost by bleeding or by lysis faster than it is made,so making the patient anaemic. The two places fromwhich blood is most often lost outside the body are thegut (intestine) and the uterus (womb). When there isbleeding into the gut, blood is found in the stools and canhe tested with the occult blood test in Section 10.10.When blood is being destroyed inside a patient, he is saidto have a haemolytic anaemia. As you read in Section1.18, haemolysis means the destroying, breaking open,or lvsis of red cells.7.6. Iron deficiency anaemiaIn many places this is the commonest kind of deficiencyanaemia. Sometimes the anaemia is due only to thepatient not having enough iron in his food. More often apatient is anaemic partly because there is not enoughiron in his food, and partly because he is losing ironbecause his body is bleeding. The commonest reasons forthis loss of blood are hookworm infection and abnormallyheavy monthly periods.As we saw in Section 7.3 iron deficiency causes thered cells to be poorly filled with haemoglobin and thus tobe hypochromic with an MCHC below 32%. As we shallsee in Section 7.19, the red cells of iron deficiencyanaemia are often smaller than normal or microcytic.Iron deficiency therefore causes a hypochromic microcyticanaemia.HookwormanaemiaHookworms are small nematodes or round worms whichlive in the small intestine (small gut). They hold on to thewall of the small intestine with their mouths. The wall ofthe intestine bleeds a little where each worm bites it.From the bite of each worm the patient will lose about &,ml of blood each day. A few hookworms will thereforecause little bleeding and no anaemia. But a heavilyinfected patient may have several hundred worms in hisgut, and thus loses much blood. With the blood he willlose much haemoglobin and thus much iron. He may losemore iron than he eats and therefore easily becomesanaemic. The importance of hookworms as a cause ofanaemia depends upon how many of them there are inthe gut. Many worms in the gut produce many ova in thestool, so by counting the ova in the stool we can get someidea about how many worms there are in the gut. This isdescribed in Section 10.3.Hookworm anaemia is common in children, so allanaemic children must have their stools looked at forhookworm ova and these ova must be counted. It is veryimportant to diagnose hookworm anaemia, because it ischeap and easy to treat. A moderately anaemic child isone who has a haemoglobin of between 5 and 10 g %. Achild with an anaemia of this kind can easily be treated ata health centre with a drug called tetrachloroethylene(‘TCE’) which will kill hookworms in his gut. Bephenium(‘Alcopar’) is also used to treat hookworm infection. Achild will also need iron to replace the iron he has lostand so help his body make more haemoglobin. Sometimesthis iron is given as tablets and sometimes as aninjection. Iron injections such as ‘Imferon’ are expensive,but they are useful because all the iron needed by thebody can if necessary be given at one time, or over a fewdays. When iron tablets or an iron medicine are given,children may not take them as they should. Childrenwith a haemoglobin of less than 5 g % are very anaemicindeed and must be treated in hospital where they canhave a blood transfusion as well as iron. Read aboutblood transfusion in Chspter Twelve.

Very heavy infection with the worms calledSchisfosoma haematobium or Schistosoma mansoni canalso cause an iron deficiency anaemia. But anaemias ofthis kind are only common in a few countries, and theremust be many worms before the anaemia is severe.Anaamia due to very heavy monthiy periodsJust as many hookworms may together cause muchblood to be lost into the gut and make a patient anaemic,so a woman who loses a lot of blood with her monthlyperiods may also become anaemic. A woman with veryheavy monthly periods is said to suffer frommenorrhagia She usually knows that she is bleeding toomuch. Like patients with hookworm anaemia, womenwho are anaemic from heavy monthly periods also neediron. But most women can be given iron tablets whichare cheaper and easier than the iron injections whichchildren often need.Although iron deficiency is the commonest cause of ahypochromic microcytic anaemia in most areas, thereare some places in which a disease called thalassaemia isthe commonest cause of this kind of anaemia-seeSection 7.27b. Thus, although most iron deficiencyanaemias are hypochromic, not all hypochromicanaemias are due to I’ron deficiency.7.7 Folic acid deficiency anaemiaIn some parts of the world this is a very common causeof anaemia, especially in antenatal and nursing mothers.Antenatal means before the birth of the child. So anantenatal mother is a mother with a child in her womb. Anursing mother is a mother who is giving milk to herchild. These mothers become anaemic because they donot get enough folic acid in their food with which theirbodies can make haemoglobin. A child in his mother’swomb (uterus) or sucking from her breast also needsfolic acid. Often there is not enough folic acid in amother’s food for both a mother and her child. Themother therefore becomes anaemic. The anaemia due tofolic acid deficiency is a macrocytic kind of anaemia andis described in Section 7.19. It is important to recognizefolic acid deficiency anaemia because it is easily andcheaply treated with folic acid tablets. Antenatal andnursing mothers also often lack iron. Their anaemia maytherefore be due to a double deficiency of iron and folicacid. Antenatal mothers are therefore often given tabletsof both iron and folic acid.Folic acid deficiency is also common in haemolyticanaemia and in anaemia due to infection. Patients withboth these kinds of anaemia may therefore be helped byfolic acid.A much less common kind of deficiency anaemia is,, that due to lack of vitamin B,,.7.8 Anaemia caused by protein deficiencyPatients with protein-joule malnutrition or ‘PJM’ oftenbecome anaemic because they do not eat enough proteinwith which their bodies can make red cells. The red cellsare often slightly larger than normal as well as beinghypotihromic.7.9 Haemolytic anaemiasThis kind of anaemia is due to red cells being destroyedor lysed inside the body. In areas where malaria is seen,this is the commonest cause of a haemolytic anaemia,especially in children between the ages of 3 months and 5years. This is because, when malaria parasites growinside red cells, they destroy them, and the patientbecomes anaemic. When a patient’s anaemia is due tomalaria, parasites can usually but not always be seen in athick or thin blood film, as described in Section 7.3 1. Ifmalaria parasities are seen in large numbers it is probablethat they are causing most of his anaemia. If thereare only small numbers of malaria parasites in the bloodof an anaemic patient there is likely to be some othercause of the anaemia also. So, don’t be satisfied by diagnosingmalaria alone as the cause of anaemia, especiallyin a child, without looking for other causes also. Whenthe anaemia is due to malaria you may see large numbersof mononuclear cells, as described in Section 7.14.Sickle-cell disease is another common cause of haemolyticanaemia in areas where this disease is seen (seeSection 7.24).In haemolytic anaemias, and especially in sickle-cellanaemia, polychromasia is often obvious (see Section2.27a). At the same time many reticulocytes may befound (see Section 7.23). Abnormal amounts of a substancecalled urobilinogen are usually found in the urineof patients with haemolytic anaemias-but not withanaemia due to bleeding (see Section 8.8). Finding manyreticulocytes in the blood and urobilinogen in the urineare therefore useful ways of telling if a patient has ahaemolytic anaemia.Several causes often combine to make a patientanaemic. For example, a patient’s anaemia may be partlydue to malaria, partly to hookworm infection, and partlyto a deficiency of protein.There are many other causes of anaemia, and it isoften dimcult to tell why a patient has anaemia withoutspecial methods which are not described in this book.Some other causes of anaemia, such as uraemia andleukaemia, are shown in Figure 7- 17.7.10 When to measure the haemoglobinBecause we cannot measure the haemoglobin or haematocritof all patients coming to a clinic, an outpatientdepartment, or a health centre, we have to choose onlythose patients who are most likely to be anaemic. Buthow do we know which ones they are? When any patientis examined, one of the things that must be done is tolook at his tongue, and at the red skin inside his lowereyelids-his conjunctivae. The tongue and conjunctivaeof healthy people are a deep pink colour, but in anaemicpatients they are pale. Patients with a pale tongue and

The thin blood film 1 7.11pale conjunctivae are probably anaemic and must havetheir haemoglobin measured. When you have had somepractice, it is quite easy to End anaemic patients bylooking careIully at their tongue and conjunctivae.Whenever you think someone has anaemia you mustmeasure his haemoglobin or haematocrit.It is especially important to examine children andwomen fir anaemia. Anaemia is common in young children,because they are growing fast and do not get theright things in their food. Because of this their bodiescannot make enough blood. They often suffer from hookworminfection and malaria which also make themanaemic.Anaemia is common in women because they have togive food (especially iron, protein, and folic acid) fromtheir own bodies to make their children’s blood. Theyalso lose blood with their monthly periods and at childbirth.Women are most likely to be anaemic during pregnancyor soon after delivery. Measure the haemoglobinor haematocrit of mothers four times, if possible:1. When they come to the antenatal clinic;2. When they are 32 weeks pregnant;3. When they are 38 weeks pregnant;4. When they come to the postnatal (after birth)clinic.Any patients who say they feel tired and lack energyshould have their haemoglobin measured, becauseanaemia often makes people feel tired.One more thing must be said about anaemia. If youmeasure the haemoglobin of a patient just after he hasbled a lot suddenly, you will find that the little blood hehas left will still be normal. He will not be anaemic. If,however, you look at his blood a day or two later, youwill lind that he will now have become anaemic. This isbecause he will then have had time to dilute his remainingblood, so that its volume is increased. Blood whichhas been diluted by the body in this way becomesanaemic. The body works better with a larger volume ofdiluted anaemic blood than it does with a smaller volumeof normal blood. But for the body to work in the bestway it can, and for the patient to be healthy, the anaemiamust be cured by more red cells being made.When you have found an anaemic patient, the nextthing to do is to look at a thin film of his blood.THIN BLOOD FILMS STAINED WITHLEISHMAN’S STAIN7.11 The thin blood filmAs you will remember from Section 1.17, normal bloodcontains millions of red cells and smaller numbers ofwhite cells in a liquid called plasma. It is very useful tolook at these cells with a microscope to see whether theyare normal or abnormal. If the cells are abnormal wehave to find out what is wrong with them. Before bloodcells can be looked at with a microscope they have tohe spread out very thinly on a glass microscope slide. Aslide covered very thinly with blood is called a thin bloodfilm. The cells in a thin blood 6lm cannot be seen unlessthey are first stained. The stain we use is calledLeishman’s stain. Someone who is well practised in examiningthin blood films can find out many useful thingsabout a patient’s b!ood. But it will only be possible hereto tell you a few of the more important things that can beseen in a thin blood film. First you must learn how tomake the film.The stainand the bufferRead how to make Leishman’s stain in Section 3.33 andhow to make Leishman buffer in Section 3.21. Themethyl alcohol from which Leishman’s stain is madesoon goes into the air and dries up-that is, it rapidlyevaporates. Leishman’s stain must therefore be kept in abottle with .a stopper or lid that fits tightly. It is alsouseful if the bottle has a pipette in the stopper with whichthe stain can be measured. To make it easy to do this,some special bottles, called Polystop bottles, have beenput in the equipment list (ML 7). One Polystop bottle isfor the stain and another is for Leishman buffer. Don’tput the pipette for the buffer into the bottle for the stain,because, if any water or buffer gets into the stain, it willnot work. No water or buffer must ever get intoLeishman’s stain-it must be kept dry, or free of allwater which easily gets into it from the air.A buffer was described in Section 1.7 as being a mixtureof special salts which helps to keep the acidity oralkalinity (the pH) of a solution fixed (the same). Leishmanbuffer is made from two kinds of phosphate whichare powdered, weighed, and then mixed together. A littleof the powdered mixture of salts (about 200 mg) is addedto a Polystop bottle full of water to make the buffer.When plain ‘dry’ water-free Leishman’s stain is put onthe film, the methyl alcohol in the stain fixes (kills andpreserves) the cells-see Section 4.10. When buffer isadded, the wet mixture of stain and buffer then stains thecells. Unfixed cells will not stain well and wet stain willnot fix well. This is why it, is so important to keepLeishman’s stain dry.7.12 Leishman’s methodIt may look easy to spread blood on a slide so as to makea good thin blood film. But it is not so easy as it looks.The only way to learn how to make good films is to doeverything you read here very carefully. You must go ontrying until you really can make good films. Don’t becontent with bad ones!The first thing to know is what a good thin blood filmshould look like. This is shown in Picture A, FIGURE 7-7.The film should start close to one end of the slide; itshould very nearly reach the other end and come close tobut not touch the edges of the slide. The film starts at thehead and ends at the tail. The blood cells must lie evenlyon the slide. This means that there must be no holes inthe film, and no lines across the film, or down its length.

this is a jar of spiritin which clean slides arestoredleadytobeusedthe surface of a slide isbeing rubbed clean15 la”elleaa slide is being flamed, thishelps to get rid of any greasethat may be leftItvs/is a pair of plierstn15 IS a suae wnlcn nas been chosenbecause it has a very smooth edge, thecorners of this edge are being chippedoff with a pair of pliers : this willmake a spreader which will be keptcarefullyblood can also be taken from an ear or a fingerput the spreader on the slidein front of the drop of bloodand draw it backwardswait for the blood to flowacross the end of thethis is the slide whichhas been carefully cleaned8the spreader will leave a thineven film behindmake your film quickly, don’tlet the drop of blood dry up9 don’t liftpush the spreader forwardsteadily and quickly- the date acrenumber of drops needed tocover the filmFig. 7-6 The Leishman blood film-onestaining rat k

7 1 BloodA film is always too thick near its head and is usually toothin at the end of its tail. In a good thin blood film thecells are just separated from one another towards the tailin the place shown by the dotted lines in Picture A,FIGURE 7-7. But, if the film is made too thin the cellswill be too far apart. Try to make the film of the rightthickness for as much of its length as possible. The filmsdrawn in FIGURE 7- 11 are the right thickness. The ce!lsare not touching, and yet they are not too far apart. Thepatient’s name should be written in pencil across thehead of the film, and when stained a goort thin filmshould be an even pink colour.You can only make a good film if you do exacfZj~ whatyou are told in the method which follows.METHODMAKING A THIN BLOOD FILM. FIGURES 7-6, 7-7. and 7-8I. Choose your best slides for making thin blood films;keep old scratched slides for stools. Wash the slideswell with a detergent or with soap and Water. Dry themand keep them in a Screw-Capped jar filled With spirit. ffyou still cannot get clean slides with which to makegood films, try cleaning them with dichromate as describedin Section 3.12.2. When you want to us8 a slide, tak8 it out of thespirit jar and rub it dry with a piece of clean cloth orpaper. Us8 toilet papar.3. Quickly put the Slid8 through a flame and allow it tocool. All these things help to make quite sure that theslid8 is clean and free from grease. They may not alwaysbe necessary, especially with new slides. But, if youfollow them. you will at least start with 8 slide on whichit is possible to make a good film.4. You need a slid8 to spread the blood with. This isthe spreader. Look through your slides until you find on8which has a very smooth edge, without any chips. The8dg8 of a spreader should be a little narrower than aslide. So take a pair of pliers and carefully break off thecarners of the spreader. Break off very small pieces ofglass with each bit8 of the pliers. When you have mad8a good spreader, look after it carefully. Make a newspreader every few days, beCaUS8 new spraaders se8mto make better films than old ones.5. Put the slide down on the bench. Using a wire loopor a Pasteur pipette put a small drop of fresh or sequestrenatedblood in the middle of on8 end of the slide.ClOSe t0 its edge.6. immediately hold your spreader so that it slopesbackwards. Put its spreading edge just in front of thedrop of blood.7. Move the spreader backwards so that it touchesthe drop of blood. When the blood touches the spreaderit will flow across between the edge of the spreader andthe slide. lf it does not spread, lower the spreader alittle.8. As soon as the blood has spread right across theslide, push the Spreader forward steadily and rapidly. Bysteadily we mean without stopping. The blood will thenbe left behind on the slide as 8 thin even film.9. If the drop of blood was the right size the film willend just before the 8nd of the slide in a nice thin eventail.IO. immediately pick up the slide by one end andwave it about rapidly until it is dry. This will stop the redcells forming rouleaux-see Picture F, Figure 7-l 1, andSection 12.6.11. Write the patient’s name and the date across thehead or side of the film. Us8 an ordinary pencil. Thewriting will stay if it iS Written on the film.12. Put the film flat across the two bars of the stainingrack.13. Cover the film with a few drops of Leishman’sstain, putting the drops in different places on the filmuntil the whole film is covered. This plain dry stain fixesthe Cells.14. Immediately add twice as many drops of buffer.Thus, if five drops of stain w8r8 no8d8d to cover theblood film, add ten drops of buffer. Some people waiton8 minute before adding buffer to the stain. But, if youwait too long, the methyl alcohol in the stain will evaporateand leave pieces of blue stain on the finishedSlidF+-Se8 Picture F, Figure 7-l 1.15. Because the stain is made with methyl alcohol,and the buffer is made with water, they do not mix easilyby themselves. They have to be mix8d. So, mix the bufferand the stain by sucking them in and out of the bufferpipette. Don’t use the stain pipette, beCaUSe buffer willget into the stain and make it wet. This will spoil thestain.16. Another way of mixing the stain and buffer is toblow gently up and down the slide, from on8 8nd to theother. Don’t blow too hard or you will spill the mixture.17. Wait about 5 minutes while the wet mixture ofstain and buffer stains the cells.18. Flood the stain-buffer mixture off the slid8 withmore fr8Sh buffer. Pick up th8 slide with forceps ratherthan with your fingers as shown in the pictures. Washthe slid8 with a little more buffer.19. Leave the buffer on the slides for about 3 minutes.The slide should now be a good pink colour and not blueor purple. If it is not pink 8nOUgh leave some morebuffer on it for a bit longer.20. Tip off the buffer and leave the slide upright in arack (21).22 and 23. As soon as the film is dry put a drop of oilon it and smear it all over with the rod of the oil bottlesome people use their fingers. Others put a coverslipover a drop of oil. Unless the film is covered with oil,cells will not be seen clearly.7.13 Faults in a thin blood filmLook over the film with a low power objective. This willshow you what it looks like and will let you find a goodplace to look at with the high power objective. You willseldom need to use an oil immersion objective, and

the film has beenthe stain and the buffer do not-I-_ __-:I_. L.. -L----I ..-.blow gently across the slidefrom end to endm’x pas”y OY Knem=‘ves .,-SF>&_r,cover the film with bufferand leave it for about 3a fine green scum willwhe stain and washit.away with buffer/5put a drop of oilon the slide‘bufferleave the slideto dry in a\rack I _oil on itsmear the oil over theslide \ \ Lx,this is the head of the film,it is always too thick here.--this is the place to writethe patient’s name and thedate : use a pencilOne good film and six bad oneswhen vou do a differentialA white ceil count follow this linethe film killbe the right thickness\thisis the rod orstick from the oilbottle, some peopleuse their finger,thls is the tail of the film,too thin hereGOODthere are more polymorphsalong the edge of the filmand in the tailthere are more lymphocytesin the middle of the filmto look at inside this square6 BADcD BADEthe film goes off the endof the slideFthe spreader has beenlifted up hereG "there was a chio in the soreaderthe slide wa: greasy and thereare holes in the filmthe firm is much too short and thickFig. 7-7 The Leishman blood film-twothe spreader was pushedforward jerkily..-’

Faults in a thin blood film 1 7.13THIS MAKES THE FILMTHINNER \ FITUlP MAYCC l-UC FILMI rllCl I”IriI,LU I I ILTHICKER\ STEEPUthe spreader has been speciallychosen so that it has a smoothedge and has been labelled sothat it does not get mixed upwith other slidesthisisthedipofblood whichspread outis being/yythis is the slide on which the bloodfilm is being madechippedz,;akesthere beenoff so thata narrowerFig. 7-8 Adjusting the thickness of a blood filmshould not use one for a differential white cell count. The was greasy. In Picture F the film is much too short andbest place to look will probably be towards the tail of the thick. The spreader must have been held too steeply orfilm. This area is shown with a line round it in Picture A, pushed too fast, or both. There are lines across the film inF~CURE 7-7. Here the cells should lie evenly without Picture G because the spreader was pushed along it in atouching or lying on top of one another. jerky way-stopping and starting.It is more difficult to make a good film with anaemicblood because the 6lm easily becomes too thin. Stop thisOther faults in blood films are shown in Picture F,FIGURE 7- 11. On the left-hand side is a heap of redby holding the spreader more steeply and by pushing it blood cells that have come together in rouleaux. Redfaster. The steeper the spreader is held and the faster it is cells lying on top of one another in this way are like apushed, the thicker will be the film. The flatter the pile of coins. The way to stop this happening and to makespreader is held and the slower it is pushed, the thinnerwill be the film. When a film of anaemic blood is madethicker in this way it is especially important that it besure that the cells lie evenly as in Picture A, FIGURE7- 11, is to make the film immediure!v you have placedthe drop of blood on the slide, not to make it too thick. todried quickly by being waved about, as in Picture 10, make the film thinner, and to wave it dry rapidly as SOOHFIGURE 7-6.as if is mnde. Picture F, FIGURE 7- 11, also shows whatWhen you have finished looking at the blood films for happens if you let the stain dry up on a slide before it isthe day, stand them upright on end in a box. Wipe the mixed with buffer. When this happens the dried stain islllms clean with soft or toilet paper and put a piece of seen as many little round dots. Much the same thing maypaper between the films for one day and those for the happen when the green scum that forms on top of thenext day. Make the piece of paper stick out between oneday’s films and the next and write the date on it. Whenthe box is full, throw away the oldest ones and put thenewest ones in their place. If you keep blood films likethis, you will be able to find the films you have madeduring the previous few weeks very easily. This is oftenuseful.stain is not washed off with buffer as it should be.Sometimes you will find that white cells do not stainwell. This is usually because the stain has got wet. but itmay be because the methyl alcohol wa!: wet to start with.Some kinds of methyl alcohol do not work well withLeishman’s stain.Some of the common faults in blood films have beendrawn in Pictures B to G, FIGURE 7-7. A good film isshown in Picture A. All the other films are bad ones. InPicture B the film reaches right to the end of the slidebecause the drop of blood used was too big. In Picture Cthe film ends in a sharp line because the spreader waslifted off the slide before it had reached the end. InPicture D the film looks as if it is cut into two, becausethere was a big chip in the edge of the spreader. Thereare holes in the film in Picture E. This is because the slideIf the blood has been taken into sequestrene, rememberto make the film quickly and to mix the blood beforeyou do so. The cells will spoil if they are left in sequestrenefor more than an hour before the film is made.If the film looks a little too blue, it may be made morered by washing it with more buffer. But if the film ismuch too blue it is because the buffer was too alkaline(see Section 1.7). If the film looks too red, it is becausethe buffer was too acid. When this happens make newbuffer.Finally, never blot a blood$lm. Always add stain and

7 1 Bloodbuffer to one film at a time. Don’t be content with badfilms. Go on making films until you have got one whichis good enough to stain. Bad films can always be washedand the slides used again.Leishman blood films are useful for seeing if the whitecell or platelet counts are very high or very low, fordoing a differential white cell count, for looking at redcells, and for finding parasites. These are discussed laterin this chapter. Leishman’s stain can also be used forstaining the bone marrow. Marrow is obtained by puttinga special short, sharp, strong needle called a marrowbiopsy needle into a bone and sucking out a few drops ofthick red liquid marrow with a syringe. This can be diBicultand is only done by doctors. Marrow films can bestained in the same way as blood films.Remember a medical laboratory can be judged by thequality of its thin blood films-make sure yours aregood!LOOKING AT A LEISHMAN STAINED THINBLOOD FILM7.14 Normal white blood cells (leucocytes)You wi!l .remember that normal blood contains red cells,which are round and without nuclei, and white cells andplatelets. These next sections describe the normal andabnormal kinds of cell that you will see in a film.There is only one kind of red cell (erythrocyte) innormal blood, but there are several different kinds ofwhite cell (leucocyte). These are shown in FIGURE 7-9.Some of them have been drawn again in FIGURE 7- 10which also shows some of the cells of the bone marrow.FIGURE 7-9 is a careful drawing. FIGURE 7-10 is adiagram. Both show the cells as you can see them whenthey are stained with Leishman’s stain. Cells E, F, and Gare the same in both these figures.In every hundred white cells in normal blood there areabout seventy white cells called polymorphs (this is ashort way of saying polymorphonuclear leucocytes).These cells are called polymorphs because their nucleihave many shapes (poly = many, morph = shape). Whenthey are very young the nuclei of the cells are round(Pictures A. B, C, D. FIGURE 7-10). But, as these cellsget older, their nuclei become folded and twisted. Thenucleus of mature (older) polymorphs is twistedinto several segments (pieces). The older a polymorphgrows, the more segments it has. Most of the polymorphsin normal blood have two or more segments,like the cell shown in Picture G, which has three. Afew polymorphs in normal blood are like the one drawnin Picture F, and a very few are like that shown inPicture E.All polymorphs have many granules (very smallpieces or particles) in their cytoplasm, which is pinkerthan the cytoplasm of a lymphocyte. Some polymorphshave very small purple granules-these are called neutrophilpolymorphs. It is often difficult to see the granules,because they are so small and pale. Neutrophilpolymorphs are much the commonest kind of polymorph,and when people talk about polymorphs, this isthe cell they mean. Other polymorphs have much biggerred granules in their cytoplasm-these are the eosiniphilpolymorphs or ‘eosinophils’. Sometimes you will seepolymorphs with big blue granules in their cytoplasmtheseare the basophil polymorphs or ‘basophils’ whichare rare (not common).Neutrophil polymorphs can leave the blood and gointo the tissues to fight and eat micro-organisms whichcome there. If there are many polymorphs together theymake a thick yellow liquid called pus. Polymorphs in pusare often called pus cells. Pus cells are also found inabnormal urine, sputum, CSF, and stools. When somethinglooks like pus, we say it is purulent.There are two other kinds of white cells. Both havemore rounded nuclei which are often indented. By indentedwe mean that they have a dent or a notch in them.They are sometimes grouped together as mononuclearcells because, unlike polymorphs, they have only onelobe to their nucleus (mono = one). Both have few granulesin their cytoplasm which is more blue than thecytoplasm of the polymorphs. These cells are the lymphocytesand the monocytes. There are two kinds oflymphocyte-large lymphocytes and small lymphocytes.The polymorphs and the red cells are made inthe bone marrow, but the lymphocytes are mostly madein the lymph nodes (lymph glands).The small lymphocyte is easy to recognize: it is small,its nucleus is round, or nearly round, and it has littlecytoplasm (often it appears to have none). The substancefrom which the nucleus is made, the nuclear chromatin(see Section 1.9), is dense (solid) and &eply staining. Thelarge lymphocyte is larger; it has more cytoplasm inwhich are a few small pink granules; and its nuclearchromatin is less dense. In the nuclear chromatin of bothkinds of lymphocyte you will sometimes see one or twonearly empty ‘holes’. These are the remains of similarholes or nucleoli in the cells from which lymphocytes areformed. These holes are normal in the nuclei of lymphocytes,but in other white cells nucleoli usually meanthat the cell is immature (too young) and should still bein the bone marrow. Cells are often seen which are halfwaybetween small lymphocytes and large ones. Wedon’t usually try to distinguish them. Instead, we countall lymphocytes together.A monocyte also has cytoplasm with very few granules,but its nucleus is different from the nucleus of alymphocyte. Instead of being round it is often shaped likea kidney or bean-see Picture B. FIGURE 7-9. The lymphocytehas clear blue cytoplasm, but the monocyte hascloudy or ‘smoky’ blue cytoplasm. This cytoplasm oftenhas round white holes or vacuoles in it. A vacuole is asmall space which looks empty. The nuclear chromatinof lymphocytes and monocytes is also different. Thechromatin of the lymphocyte is dense (thick) andis nearly the same all over. But the chromatin of themonocyte is more open and looks as if it is made ofstrings.

Platelets 1 7.15NEUTROPHILMETAMYELOCYTEF 9 dense (dark) MONOCYTEnuclear chromatinPlate 6these cells aresometimescalled band cellsSMALLLYMPHOCYTEH Kb @@ a@tq e a@ EmPLATELETSI Plate 1,:,;\ ,-;.. :..‘fy*

WHITE CELLFAMILYcs---: --.--_ 1\ ~~RED CELLFAMILYB-h - deep\blueTO9 LASTMYELOBI\this is the last\ cell to havenucleolicytoplasmas the red cells maturethey get smaller, theircytoplasm getsless blue and fills withhaemoglobin, and theirnuclei get smaller andfinally&appearPP IOMYELOCYTEs is the first cell toMARROWhaemoglobin starts to _appear (be seen) heremany granulesce:ME LTAMYELOCYTEFBAND CELLas the white cells mature,they get smaller, theircytoplasm getsless blue, granules formin the cytoplasm and thesmall dark roundnucleus becomesnucleusless round andmore foldedTHE CELLS ABOVE THISr-LINE SHOULD STAY IN -THE MARROWBLOODthis cell is a purplecolour when stainedwith Leishman’s stainLLYCHROMATICD CELLPlate 44MATUREPOLYMORPH-ate 2nse nuclear chromatinof these areents or piecese nucleusFig. 7-10 How the blood cells are formed in the marrowMTUREPlate 1RED CELLmature red cell isa red colour whenstained withLeishman’s stain

How blood cells are formed in the marrow 1 7.17person and count the first hundred white cells you saw,they might be like this:Neutrophil polymorphs 70Lymphocytes 25Monocytes 3Eosinophils 2You might or might not find a basophil, because basophilsare rare. This is blood from a normal person, andthis percentage of the different kinds of cell (this numberof white cells in every hundred) is what we mean whenwe say a person has a normal diierential white cellcount. The normal differential white count is not alwaysexactly like this, and in adults we usually take any figurewithin those shown below as being normal.Neutrophils 40-75%Lymphocytes 20-45%Monocytes 2-10%Eosinophilsl-6%Basophils Less than 1%For example, a film with only 5% of neutrophils or afilm with as many as 20% of eosinophils would not benormal. It is to find out things like this that we do adifferential white cell count.Normal people have difBerential white counts withinthese figures. They also have no abnormal red cells orabnormal white cells in their blood. To understand theseabnormal cells, you must first learn how cells are formedin the marrow. When you have learned a little moreabout white cells, you will be able to do a differentialwhite count. This is described in Section 7.22.7.17 How blood cells are formed in the marrow(FIGURE 7- 10)New cells have to be made all the time to replace oldcells which wear out in the blood as it goes round thebody. Polymorphs and red cells are both made in the redmarrow, which is inside some of the bones. Only whencells are fully mature should they leave the marrow andgo into the blood. You will see this red marrow if youlook at animal bones which have been cut up in abutcher’s shop. Marrow is the soft red substance in themiddle of a bone. If we look at this marrow under themicroscope we see that it is made of cells of many differentkinds. The less mature of these cells can divide into(make or cut themselves into) two daughter cells. Becausemany cells in the marrow can divide in this way,there are always more cells to come into the blood. Theimmature cells which can divide all have big round nucleiand little cytoplasm and stay in the narrow. It is theirmature daughter cells which leave the marrow to goround the body in the blood.The cells of the marrow have been drawn in FIGURE7-10. At the top of the picture is the parent cell(haemocytoblast). This divides to make polymorphs asshown on the left and red cells as shown on the right. In ahealthy person all the cells above the line stay in themarrow, and only those below the line leave the marrowand come out into the blood. The haemocytoblast is alarge cell with a big nucleus and little cytoplasm. As cellsmature they get smaller and their nucleus gets muchsmaller and more solid looking. At the same time theircytoplasm gets less blue. Cells which are going tobecome red cells lose their nucleus altogether, andhaemoglobin forms in their cytoplasm. Thus cell Achanges into cell H, then into cell I, and so on until themature red cell M is formed. Cell I is the first to havehaemoglobin and therefore stains pink. It is an earlynormoblast. Cell J is in between the two, and is anintermediate normoblast. Intermediate means in themiddle.Similarly, as they mature, the nucleus of the cells thatare going to become polymorphs gets narrower as wellas smaller. The nucleus soon gets so narrow that it twistsor cuts into segments, and the mature segmented polymorphG is formed. The promyelocyte C is the first cellto have granules and the last cell to have nucleoli.FIGURE 7-10 only shows how red cells and neutrophi1polymorphs are formed. But eosiniphils and basophilsare formed in just the same way, and there areeosinophil and basophil cells like cells C, D, E, F, and G.7.18 Abnormal cells in the bloodSo far we have only talked about the normal cells in theblood. In sick people many kinds of abnormal cells areseen in the blood. Cells can be abnormal in two ways.They may be abnormal because they are badly made. Weshall describe several kinds of badly made cells. Cellscan also be abnormal because they are too immature (tooyoung) to be in the blood. As you see in FIGURE 7- 10the immature cells above the line in this figure shouldstay in the marrow. They are abnormal if they are foundin the blood. Some cells are both badly made and immature.We shall first describe abnormal red cells and thenabnormal white cells.7.19 Abnormal red cells (FIGURE 7- 11)The normal red cells of a healthy person are all nearlythe same size (7.5 jrrn across) and they are almost perfectlyround. They have no nucleus. When stained withLeishman’s stain, they are a good pink colour with only asmall pale area in the middle. As we have already seen,normally coloured red cells like this are said to be normocbromic(normally coloured). They have been drawnin Picture A.We shall first describe abnormalities due to the cellsbeing badly made and then abnormalities due to the cellsbeing immature.Badly made red cells The red cells of anaemic patientsare often of different sizes. Some cells are small andsome are large. When red cells are of different sizes likethis, they are said to show anisocytosis (unequal-sizedcells) and are shown in Picture B. Anisocytosis is commonin many blood diseases.

me Iypochromicare different sizes, many are small,‘$some are not round and they look i’oi-Zpale and empty in the middle~“~>.microcyte,,cFi:~, 6:$I .j ~~&;~2iye.+.:>S ..:2.7...:.t!pale empty middle :mSICKLE CELL ANAEMIAl’:.’: : tI‘;. ..;z,_._,.‘.Z,.>. : .. 4.: .:.y;$..!. ._ ...‘..’ ::r,.. . i ...r.;;.,,*.._.;.&(ru>;+ygf;..:.r:-~’this is a late normoblast -@‘j6%@DPARASITESEorreliaduttoni\.;: -2

The red cells of anaemic patients are often of manydifferent shapes instead of being roundRed cells ofdifferent shapes are said to show poikRocytosis, and areshown in Picture D. If poikilocytosisis mild, the redcells are just not quite round. If poikilocytosis is severe,the red cells may have very strange shapes indeed. If yousee poikilocytoses, report it.Another kind of badly made red cell is the target cell.These are cells which are thickened in the middle and aresaid to look lie a target. A target is a piece of cardboardwith a spot on it which people shoot at with guns to seehow weU they can shoot. A thin film showing both targetcells and sickle ceUs is shown in Picture D. Target cellsare often seen in sickle-ceU anaemia.Often the red cells of anaemic patients are too pale,because they have too little haemoglobin in them. Theyhave large pale areas in the middle, and there is onlyenough haemoglobin to make a thin ring around the edgeof the cell. This is usually due to the patient not havingenough iron to make enough haemoglobin to fiU his redcells. As we saw in Section 7.3, patients like this are saidto have a hypochromic anaemia. By this we mean ananaemia with an MCHC of less than 32%. As well asbeing partly empty of haemoglobin the cells of suchpatients are also usually smaller than normal. Thesesmall red cells are called microcytes (micro = small).Because hypochromic anaemias can be so easilytreated with iron, it is important to recognize them. But,if hypochromia is to be recognized in a blood jilm, thefilm must be weII made. But even if the film is weU made,it may not always be easy to judge hypochromia,especially if it is mild. This is why the MCHC isso useful.There are some kinds of anaemia in which the red cellsare larger than normal, but there are very few of them.These large cells are called macroeytes (macro = big)and these are the macrocytic anaemias. The cells are bigand full of haemoglobin, so they stain a good pink colourand are not small and pale, like the cells from thehypochromic microcytic anaemias. A film from a patientwith macrocytic anaemia is shown in Picture C. In somemacrocytic anaemias a special kind of immature red cellmay be seen in the bone marrow and sometimes in theblood. This is the megaloblast. The nucleus of a megaloblastis much less mature than its cytoplasm. Forexample, you may see a cell in which the cytoplasm isfull of haemoglobin, but the nucleus may be like that ofanearly normoblast which has no haemoglobin. Large polymorphswith many segments and sometimes Howell-Jolly bodies (see below) are also seen in the blood filmsof macrocytic anaemia. These anaemias may be due to adeficiency of folic acid, or less often to a deficiency ofvitamin B,,. Macrocytic anaemias due to folic acid deficiencyare very common in countries where people’sfood contains too little folic acid. Vitamin B,, deficiencyis seen all over the world, but it is rare. The blood filmsof folic acid and vitamin B,, deficiencies look the same.One way to distinguish these deficiencies is to test thegastric juices for free acid, as is described in Section11.9. There is never any free acid in the gastric juice ofpatients with the disease called pernicious anaemia whichis due to vitamin B,, deficiency. Thus, if free acid isfound, this disease can be ruled out.Immature red cells. Late normoblasts are often seen inthe blood of patients with severe anaemia. They look likenormal red cells except that they have a small dark nucleus.A normoblast of this kind if shown in Picture D.Before it matures to become a normal red cell, a normoblastchanges into a cell called a polycluomatic redcell. ‘Poly’ means many, and ‘chromatic’ meanscoloured. When stained by Leishman’s stain a polychromaticred cell is purple. The purple colour is made by theremains of the nucleus dissolved in the cytoplasm. Becausepolychromatic red cells contain the remains of thenuclear chromatin, polychromatic red cells are young redcells.When a young red cell is stained with a stain calledbrilliant cresyl blue, the remains of the nuclear chromatinare seen as a deeply staining blue net, and the cellis called a ceticulocyte (net cell). Poi’ychromatic red cellsand reticulocytes are ‘the same cells stained in dIrerentways. Both a reticulocytosis (many reticulocytes in theblood) and polyc@omasio (many polychromatic cells ina Leishman-stained blood film) mean that the blood containsmany young red cells. They mean that the marrowis busily making many young new red cells. This isusually to replace those that have been lost by bleedingor haemolysis. A few normoblasts are also seen whenthere is severe polychromasia or reticulocytosis. This isbecause, when the marrow is pushing many youngred cells into the blood very fast, it pushes out a fewvery young red cells indeed-those red cells whichare so young that they still have whole nuclei (normoblasts).Polychromatic red cells are not usually counted (wecount reticulocytes instead), but you must always reportthat you have seen them in a film. A normal blood filmhas very few polychromatic red cells. If you find polychromaticred cells, be sure to report them, because thisis of help in diagnosing a haemolytic anaemia.Sometimes, particularly in sickle-cell anaemia and infolic acid deficiency anaemias, you will see red cells withwhat looks like a very small nucleus in them. This is theremains of the nucleus and is called a Howell-Jolly bodyafter the people who first described them. There is aHowell-Jolly body in Picture C. A red cell with aHowell-Jolly body in it is both young and abnormal.They are not important, but as you will probably seethem you should know what they are.7.20 Abnormal white cellsAbnormal lymphocytes and monocytes are seen, butthey are not common, and we will not describe themhere. Several kinds of ‘abnormal polymorphs’ are seen.Most of them are abnormal because they are too young.What it means to find these abnormal white cells in theblood is described in the next section.

this is the youngest and mostimmature cell in the marrowfrom which the othersare formed, it is also shownas cell A in Figure 7-10, these ‘holes’ in theof very young cellscalled nucleolinucleusarethere are several intermediate cells here,which have not been drawn : they are thecells shown H, I, and J in Figure 7-10Plate 16b~~~nucleated red cell (normoblast]this cell is stained withLeishman’s stainthese are the same cellsdifferently stainedthis cell is stained withbrilliant cresyl blueFig. 7-l 2 PolychromasiaMetamyelocytes and myelocytes are ‘young polymorphs’.They have cytoplasm containing the same kindof granules (neutrophil, eosinophil, or basophil) asmature polymorphs. But the nucleus of a myelocyte isround or nearly round, and the nucleus of a metamyelocyteis kidney- or bean-shaped. The nucleus of a myelocyteis the same shape as the nucleus of a monocyte,but its chromatin is more dense (thicker and darker),You can easily tell them apart by looking at their cytoplasm.The cytoplasm of a monocyte is quite differentitis a smoky blue with hardly any granules in ir.Promyelocytes and myeloblasts are even youngercells. Neutrophil promyelocytes have a clear or very paleand reticulocytesblue cytoplasm and very few purple granules. They haveholes in their nuclear chromatin (nucleoli). Myeloblastshave large round nuclei, no granules, and very clearnucleoli.In macrocytic anaemias, you will sometimes see largebadly made polymorphs with many segments. One ofthese has been drawr, in Picture C. FIGURE 7- 1 1.7.21 Some further blood picturesMany diseases have special blood films, or ‘blood pictures’as they are sometimes called. Several of these havealready been mentioned. Here are a few more.

The differential white cell count 1 7.22Oisaases caused by bacteriaMany bacteria, when they multiply in the body, cause themarrow to produce many neutrophil polymorphs. Theremay perhaps be 15,000 or 20,000 white cells per cu mm(see Section 7.16 for the normal values). Most of thesewill be neutrophil polymorphs. Apart perhaps from a fewmetamyelocytes, the cells will not be immature. This is apolymorpb leucocytosis. One of the main uses of a diierentialwhite cell count is to see whether or not a patienthas such a polymorph leucocytosis. If there is a polymorphleucocytosis, the patient probably has a bacterialinfection. Patients with whooping cough often have aleucocytosis (perhaps 40,000 cells or even more).Whooping cough is caused by a bacterium, and the cellscausing the leucocytosis are lymphocytes.Infectr’ons with parasitesMany kinds of parasitic worm may cause the marrow toproduce many eosinophil polymorphs. Sometimes 20%of the white cells in a blood film may be eosinophils. Ifyou find an eosinophilia like this the patient is probablyinfected with parasitic worms. In malaria the proportionof mononuclear cells may be increased.LeukaemiasThese are rare diseases which make patients anaemic andoften kill them within a few months. Sometimes you willsee patients with very many white cells-perhaps100,000 or more. These may be lymphocytes (lymphaticleukaemia) or cells from the marrow (myeloid leukaemiaor marrow leukaemia). In leukaemia many of the cellsmay be very immature. Thus, in myeloid leukaemia, aswell as seeing mature polymorphs, you will see myelocytes,promyelocytes, and myeloblasts. There are twomore kinds of leukaemia-acute leukaemia (lymphaticor myeloid) and chronic leukaemia (this again may belymphatic or myeloid).7.22 The differential white cell countNow that you have learned how to make a Leishmanstainedblood film and how to recognize the blood cells.you will be able to do a differential white cell count. Inthe method described below we include any nucleatedred cells we see, as well as the white cells. This is convenient,bo ; some people prefer to report having seen somany nucleated red cells for every hundred white cellsthey have counted.METHODTHE DIFFERENTIAL WHITE CELL COUNT. FIGURE 7-13Make a blood film and stain it with Leishman’s stain.Take a piece of paper and write down underneath oneanother the words: neutrophils, lymphocytes, mono-cytes, and eosinophils. If you come across any othercells write the names of these down also.Find a place on the edge of the film where the cellsare spread out evenly as shown in Picture A. Figure 7-7.With most microscopes it is best to look at the film withthe high power objectives and the x10 eyepiece, but insome microscopes it may be necessary to use the oilimmersion objective. Even if your microscope is onewith which it is possible to use the high power objective,you may find it useful to have a closer look at a few ofthe cells with the oil immersion objective.No&+ilS #I+#+#tt +I)+- -tlbwlwH+wt+ * 50L7++3+~s HH fl# -tM #It SRI- 1sMm+ ftw+RtII 1%munt the cells in blocks of fiveE osctao & w------make we that they add up to 100 :the numbersof each kind of cellthat YOU have counted will thengive YOU the percentage withoutany calculationwith four up and down strokes Sand one stroke acrossuse an odd piece of paper tocount the cells onFig. 7-l 3 A differential white cell countFor each cell of a particular kind you see put a strokeon the paper. When you have got five cells of a kind, putthe fiih stroke across the other four. Start in a separateplace when you come to the sixth cell and the sixthstroke. In this way you count the cells in groups of five.This will make it easier to add them up at the end. Whenyou have counted the cells in one field move on to thenext one. Move across and down the slide as shown inPicture A, Figure 7-7. It is important to move across theslide like this because there are more lymphocytes inthe middle of the film and more polymorphs along theedge. If you are going to get a true count you must lookat as many fields in the middle of the film as you doalong its edge.When you have got 100 cells, count up the numberof each different kind and report them as a percentage.It is better to count 200 cells and divide the answerby two.Find a place where the red cells are evenly spaced.and look at them carefully. Report any abnormalities. Ifyou see any malaria parasites, trypanosomes, or microfilaria,be sure to report them. Try to find the speciesof malaria parasite as explained in Section 7.33. Last ofall look at the platelets. If you always look at the plateletsin a blood film you will soon be able to recognizewhen there are fewer platelets than there should be.lo

7 1 Bloodstrong wire handleheat the wire beforecoverslip.D .Fig. 7-14 Staining for reticulocytesthe edge of the slide hasbeen ringed with Vaselineand paraffin waxThey may be very irregularly distributed. By this ismeant that there may be many in one part of a film, butfew in another.7.23 ReticulocytesEarlier on you read that in a Leishman-stained blood filmwe sometimes see purple or polychromatic red cells.These, we said, were the very young red cells. Reticulocytesare only these young polychromatic red cellsstained in another way. They are stained with a staincalled brilliant cresyl blue while they are wet under acoverslip. The remains of the nucleus, which was spreadall over the polychromatic red cell with Leishman’s stain,looks like little bits of purple string when stained withbrilliant cresyl blue. These purple strings or threadsoften look like a net. This gives the cell its namereticulocytemeans ‘net cell’. When a patient has toomany reticulocytes he is said to have a reticulocytosis.Like polychromasia, a reticulocytosis means thatthere are many young red cells in the blood and that thebone marrow must be working very hard to make them.In other words a reticulocytosis means that the marrowis very active (busy). Most patients who have a normalhaemoglobin have a marrow which is only ordinarilyactive. They do not. therefore, have a reticulocytosiseither. If an anaemic patient has a reticulocytosis itmeans that, even though his marrow is very active, hestill does not have enough blood. This can either bebecause his body is losing blood to the outside (bleeding)or because his blood is being destroyed inside hisbody-a haemolytic anaemia. A reticulocytosis, or poly-chromasia, usually means that either an anaemic patientis bleeding or that he has a haemolytic anaemia.METHODTHE BRILLIANT CRESYL BLUE METHOD FOR RETICULOCYTES,FIGURE 7-141. Put a drop of saline on a slide.2. Take a loopful of blood and mix it into the saline.3. Take a loopful of brilliant cresyl blue solution andmix it into the saline and blood.4. Put a coverslip on top of the drop of saline, blood,and brilliant cresyl blue.5. Make a little cup out of the lid of a tin, and make ahandle for it out of thick, bent wire. Bend another shortpiece of thick wire. A paper clip can be used. Into thedish put about equal amounts of candle wax and vaseline(Vaseline is a medical grease). Heat the dish sothat the wax and Vaseline melt (become liquid). Heatthe thick bent wire. Dip the hot wire into the meltedVaseline-wax mixture. Put the end of the wire along theedge of the coverslip. Some of the melted wax mixturewill come off the wire and go solid along the edge of thecoverslip. Do the same to all four edges of the coverslip.In this way the blood under the coverslip will be completelyclosed or sealed. The wax holds the coverslip andseals the mixture underneath. Sealing a coverslip isuseful for several other methods besides this one.Here is another method, which takes slightly longer,but which makes a dry stained film that can be kept. Thiswas the way in which Plate 17 was made.

What ahaemoglobinopathy is 1 7.24METHODANOTHER WAY OF STAINING RETICULOCYTESDissolve 3 g of sodium citrate in 100 ml of water.Take 20 ml of this solution and add it to 80 ml of saline.Dissolve 1 g of brilliant cresyl blue in the mixture. Thiswill give you 1% brilliant cresyl blue in a solution ofcitrate-saline.Put three drops of this solution in a Kahn tube. Addbetween six and twelve drops of the patient’s bloodfrom a sequestrene bottle. The number of drops of bloodto be added depends on how anaemic the patient is. Ifhe is very anaemic add twelve drops. If he is notanaemic. add only six.Mix and leave the tube in a warm place (37’CI for 20minutes.Mix by gentle shaking. Make a blood film in the usualway and look at it unstained. Reticulocytes will be seen,like those in Plate 17.The blood of a normal adult has less than 2% reticulocytes;a normal child has up to 5%. We are not interestedin a patient having too few reticulocytes but only inhis having too many-a reticulocytosis. With practice itis possible to see if there is a reticulocytosis by looking atthe film, but reticulocytes can also be counted. To do thisit is important that there are not more than about fiftyred cells in an oil immersion field. When there are toomany red cells it is difficult to count the reticulocytes.Count all the red cells (including the reticulocytes) in thesame field. Count at least 200 cells altogether and look atas many lields as are necessary. Then do this sum:Total number of reticulocytes seen x loo =Total number of red cells seenPercentage of reticulocytesSICKLECELLS7.24 What a haemoglobinopathy isWhen the red cells of some patients are kept withoutoxygen for some hours they change their shape. Insteadof being round and slightly empty in the middle, theybecome sharply pointed and are called sickle cells. Theyare called this because one of the many strange shapesthey form is said to look like a sickle. A sickle is acurved knife that is used in some countries for cuttinggrass. A sickle cell is shown in Picture D. FIGURE 7- 15.Cells sickle because they have in them a special kind ofhaemoglobin called haemoglobin S (S for sickle). Thishaemoglobin is different from the haemoglobin of normaladults which is called haemoglobin A (A for adult).When we examine blood for sickle cells we are lookingfor haemoglobin S. When someone has haemoglobin S inhis blood, we say he has sickle-cell trouble. There aretwo kinds of sickle-cell trouble, a mild kind called thesickle-cell trait, and a severe kind called sickle-cellanaemia.Haemoglobin S is inherited. That means that it ispassed from parent to child in the same way that a childis said to look like his parents. Half a child’s haemoglobinis given him by his father, and half by his mother.A normal child gets haemoglobin A from both his parents,and his haemoglobin is said to be AA. Sometimesonly one parent gives a child normal haemoglobin A, andthe haemoglobin given him by his other parent is theabnormal haemoglobin S. When half the child’s haemoglobinis haemoglobin A and half haemoglobin S thechild has the mild kind of sickle-cell trouble called thesickle-cell trait. We say his haemoglobin is AS. In manyAfrican countries about one person in five has the sicklecelltrait; so it is very common. If both parents havethemselves‘got the sickle-cell trait, they sometimes (onequarter of the times) both give the child haemoglobin S.All the child’s haemoglobin will then be abnormal andwill be haemoglobin S. We say his haemoglobin is SS.These children are often ill and anaemic, and many ofthem may die while they are still young. This diseaseis called sickle-cell anaemia. It is much less commonthan the sickle-trait-about 1% of all births in areaswhere 20% of people have the sickle-cell trait.Diseases due to inherited abnormal haemoglobins arecalled haemoglobinopathies. There are many kinds ofhaemoglobinopathy, but sickle-cell disease is the m&timportant. Cells containing some abnormal haeqoglobins,and especially haemoglobin S, may not live longin the blood and soon break or lyse. When many cellslyse the patient becomes anaemic. Abnormal haemoglobins,and particularly haemoglobin S, are thus importantcauses of haemolytic anaemia. Haemoglobin C isthe cause of another haemoglobinopathy, which iscommon in parts of West Africa.7.25 Sickle cellsAs you have read, sickle cells got their name from theirsickle shape. But red cells containing haemoglobin S mayshow many other abnormal shapes besides that of asickle. One of these is holly leaf shape. Holly is a treewith sharply pointed leaves. There are many other shapeswhich have no names: two of them have been drawn inPictures F and G, FIGURE 7- 15. Together all theseshapes are called sickle cells. The important thing toremember is that all sickle cells have long, sharp points.Sickle cells must not be muddled up with crenated redcells. Crenation is described in Section 1.18 and meanscrumpled, shrivelled, or folded up. All red cells willcrenate (whether they come from a sickle-cell patient ornot) when the plasma or saline in which they are lyingstarts to dry up. When this happens the salt in the salineor plasma around the red cells gets too strong(hypertonic) and sucks the water out of their cytoplasm.As this wate; comes out, the cells shrink (get smaller),but the red cell membrane stays the same size. Just as abig coat crumples and folds on too small a person. so thered cell membrane crumples and folds when the inside ofthe red cell gets smaller. Any red cell can crenate in thisway. Sickle cells have long sharp points, but crenated

7 1 BloodA NORMALRED CELLthis is a normal redcell looked at fromon topC CRENATEDREDCELLPlate 10mthis is a normalcut acrossred cellthese pointsare BLUNTALL red cells crenatewhen the saline in whichthey are lying gets strongerbe careful to distinguishCRENATION from SICKLINGwhen they are kept awayfrom the airFig. 7-l 5 Crenation and sicklingcells have many short points. Once you have seen themboth you can easily tell sickle cells from crenated cells.We can make red cells sickle by putting someundiluted blood from a sickle-cell patient under a coverslip,and sealing the edges with wax. This will keep outthe air, the cells will use up the oxygen under the coverslip,and the following morning sickle cells will be seen.But a whole day is a long time to wait, and it is helpful tomake cells sickle more rapidly by putting a chemicalunder the coverslip with the blood. The chemical has theeffect of using up the oxygen under the coverslip.Chemicals which use up oxygen like this are called reducingagents. Chemicals which can add oxygen arecalled oxidizing agents. If we use a reducing agent, suchas sodium dithionite, sickling may be seen in as short atime as 20 minutes. Several chemicals may be used asreducing agents, but sodium dithionite (also calledsodium hydrosulphite) is probably the best. Sodiummetabisulphite can also be used.There are some difficulties in using sodium dithionite(or metabisulphite). It must be kept dry. so the lid of thebottle in which it is stored must be kept tightly shut. Alsothe solution must be made up fresh each day, because asolution older than this may not work. If you are notsure if your dithionite is working, dissolve some in alittle water and see if it will decolorize litmus paperwhich is left in it for a few minutes. Another way oftesting it is to add a few drops of Lugol’s iodine to waterand to see if a little of your dithionite will decolorize this.If the dithionite at the top of the bottle does not work,remove the top layer and see if it works a bit lower down.If you are using sodium metabisulphite, look after it andtest it in the same way, if you think it may not beworking. Best of all, do a control test (see Section 1.24)with the blood of someone whom you know has sicklecellanaemia, dr the sickle-cell trait.METHODTESTING FOR SICKLING WITH A SOLUTION OF SODIUMDITHIONITE (OR METABISULPHITE)Make sure that the sodium dithionite that you aregoing to use has been stored dry. Weigh 500 mg (0.5 g)of sodium dithionite powder into a measuring cylinder.Add 25 ml of water. This will make a 2% solution. USETHIS SOLUTION FOR ONE DAY ONLY.Put a small drop of blood in the middle of a cleanmicroscope slide. Add to it two drops of 2% solution ofsodium dithionite that has been freshly made that day.Stir the mixture with the corner of a slide. Put a coverslipover the mixture, seal it with Vaseline and paraffinwax, and wait for 30 minutes. Lower the condenser tocut down the light and look for sickle cells with a highpower objective. If you find no sickle cells look again 30minutes later. Keep the slide in a warm place while youare waiting. If you can, keep it at 37’C (body temperature)in an incubator (a special warm box). With practiceyou will be able to find sickle cells with the low power(x IO) objective and need only use the high power(x 40) to make quite sure. When looking for sickle cellsI

Two solubility methods for haemoglobins A and S 1 7.26be sure to look along the edges of the coverslip whereslight drying often helps the sickling. Don’t muddlesickle cells with crenated cells!Here is another way of looking for sickle cells inwhich bacteria from the stool use up the oxygen under acoverslip.METHODTESTING FOR SICKLE CELLS WITH BACTERIA FROM THESTOOLFill a test tube half full of saline. Add to it a piece ofnormal stool about the size of a pea (about a gram). Mixthe stool in the saline. Let the mixture stand for a fewminutes until the supematant fluid is nearly clear. Put adrop of the supematant fluid on to a slide and mix in aloopful of blood. Cover the mixture with a coverslip, andeither seal it with Vaseline and paraffin wax or leave it ina tin containing some damp paper with the lid shut.Wait for half an hour and look for sickle cells. If the testis negative, look again two hours later. This is not such agood test as the one using sodium dithionite, becausebacteria in the stools sometimes haanlolyse red calls.There are several difficulties with these methods forshowing sickle cells. The blood must be fresh, and itmust not be allowed to dry up too much under the coverslip.The coverslip should thus be sealed. The concentrationof the sodium dithionite must not be Inore than 2%,it must have been freshly made up, and the powder musthave been stored dry. Sickle cells must also be carefullydistinguished from crenated red cells and from poikilocytes.The test also occasionally gives false positiveand false negative results, especially in the first few weeksof life (as may the next test also). More importantly thesickle cell method does not tell blood which is AS fromblood which is SS. That is, it does not tell the blood ofpatients with the sickle-cell trait from the blood ofpatients with sickle-cell anaemia.It is for some of these reasons that the next methodsare now being used which work in quite a different way.7.26 Two solubility methods for haemoglobins Aand SIf we add normal blood containing only haemoglobin A(AA blood) to a strong phosphate buffer containing areducing agent, such as sodium dithionite, the reducedhaemoglobin A will dissolve in the buffer and make adeep purple solution. Because the haernoglobin A issoluble, the solution will be clear, or nearly clear. Wecan see through a tube of the solution well enough toread a newspaper through it. Also, because the haemoglobinA is soluble. we cannot remove it by filtering thesolution through a filter paper. When we try to do this,the reduced haemoglobin A comes through the paper andcolours the filtrate a deep purple.But, if we do the same thing to the blood from a sickle-cell anaemia patient (SS blood), we find that the reducedhaemcglobin S will not dissolve in such a buffer. Itmakes a turbid solution that we cannot see through orread a newspaper through. Because reduced haemoglobinS is insoluble and makes a precipitate in this buffer, wecan remove it by filtering the solution through a filterpaper. The insoluble haemoglobin S remains on the filterpaper as a dark purple precipitate, and the solution comingthrough the paper is a clear yellow (or only the veryfaintest pink).If the blood that we add to the buffer contains somehaemoglobin A and some haemoglobin S (AS blood froma patient with the sickle-cell trait). the haemoglobin S inthe blood will make the solution turbid and we will notbe able to read a newspaper through it. When we filterthe solution, the haemoglobin S will be left behind on thepaper, and the haemoglobin A will come through andstain the filtrate pink.The insolubility of haemoglobin S can thus be used intwo ways. We can see if a mixture of blood and workingsolution is clt:ar or turbid. We can also filter it to seewhat colour the filtrate is. It is useful to do the turbiditymethod first as a screening (searching) method. If thesolution is turbid and we cannot read a newspaperthrough a tube containing it’, we know that the patient hassome haemoglobin S in his blood. He is either AS or SS.We then find out which of these he is by doing thefiltration method.Many of the patients on whom we want to do thesemethods are anaemic with a low haematocrit (packed cellvolume or PCV) and a low haemoglobin. Anaemia spoilsthese methorls so we start by centrifuging a tube of thepatients blood and, if necessary, pipetting off enoughplasma so as to leave an equal volume of plasma andpacked cells. By doing this we concentrate the red cellsand correct or make up for the patient’s anaemia.There is no picture of the turbidity method and onlythe filtration method is shown in Figure 7- 16.METHODTHE TURBIDITY SCREENING METHOD FOR HAEMOGLOBIN S1. Centrifuge the patient’s blood. Take the tuba out ofthe centrifuge being careful not to disturb the deposit ofpacked cells. With a Pasteur pipette carefully removesome of the plasma so that there is an equal volume ofplasma and packed calls left in the tube. If the patient isnot anaemic and has a normal packed call volume. youwill not need to remove any plasma. But, if ha is veryanaamic, you may have to remove quite a lot of plasmsfor there to be left an equal volume of plasma andpacked cells. By doing this we are making sure that allspecimens start this method with a more or lass normalpacked call volume, and that anaamia is corrected for.2. Make up the working solution as described inSteps 1 and 2 below.3. Pipette l-9 ml of the working solution into a Kahntuba.

weigh out 0.1 g ofsodium dithioniteon a watch glass2 ml pipette/./addlomlofbuffer for the &haemoglobin/ to a Kain tubesolubili-tymethod seeSection3.21b - MAKE FRESHWORKING SOLUTION \EACH DAYI-.bloo~&mte -centrifur the specimen andadjust the PCV to 50% byremoving excess plasmaA add 0.1 ml of bloodfilter through 5.5 cm Whatman No. 1paper - cut a larger paper down tothis size if necessary6 ’!=I , ‘: i@7clear yellowhaemoglobin SS4 m 0.1 ml of hloco has beeni 1 added to 1.9 isll of workingsalutionWhatman No.1 is‘ordinary’ filter paperpJlook at the filtratet=l f=pink deep redj‘Ihaemoglobin AS haemoglobin AA(or SCI I Ilook at the filtrates again the fol!owingFig. 7- 16 The filtration method for haemoglobin S4. Add 0.02 ml of blood. Note that this is a smallerquantity than the 0.1 ml of blood needed for the nextmethod.5. Put the Kahn tuba containing the mixture of bloodand working solution in front of the ordinary writing of anewspaper. Can you read the writing through the purpletube of solution?dayr‘If the solution is a clear purple and you can readthrough it, there is no haemoglobin S in the specimen,and the patient’s haemoglobin is AA. The filtrationmethod below is not needed.If you cannot read a newspaper through the solution,because it is turbid, some haemoglobin S is present, andthe patient is either SS or AS. It is not possible with thismethod tc tell which of these he is and you will have togo on to the filtration method.This method occasionally gives false positive resultswhen the patient has abnormal plasma proteins (dysproteinaemias),but this is rare.If the mixture is turbid by the above method, find outif the patient has haemoglobin AS or SS like this.METHODTHE FILTRATION METHOD FOR HAEMOGLOSINS AA, AS, ANDSS. FIGURE 7-16If you have not already done so, centrifuge the specimenand remove excess plasma to correct for anaemiaby doing Step 1 above.1. Weigh out 0.1 g of sodium dithionite into a universalcontainer. A small test tuba marked at the O-1 g levelwill save you the need to weigh the sodium dithioniteeach time.2. Add IO ml of the ‘buffer for haemoglobin solubilitymethods’ (see Section 3.21b) to the container with thesodium dithionite in it. Mix the dithionite with the bufferuntil it is dissolved. This is the working solution and isenough for five testa. Be sure to make up this solutionfresh each day. If there is any over at the end of the day,throw it away. This working solution is also used for theturbidity method above.3. Pipetta 1.9 ml of this working solution into a Kahntube.4. Add 0.1 ml of the patient’s blood that you havecorrected for anaemia, to the working solution in theKahn tube.5. Mix well by holding the tuba between your fingerand thumb, and turning the tuba upside down ten times.The dithionite will turn the haemoglobin in the blood adeep purple colour. Specimens with haemoglobin S inthem will become turbid from the haemoglobin that hasprecipitated in the buffer.6. Filter immediately through a 5.5-cm Whatman No.1 filter paper. If your paper is larger than this. cut itdown to this size. Too large a paper will soak up toomuch of the solution. It may also be useful to cut downone or two of your plastic funnels to fit’this size of paper.If you have difficulty in finding somewhere to stand yourKahn tubas, stand them in a lump of plasticine.7. Look at the colour of the first millilitre of the filtrate.Filtrate deep purple with nodark red deposit on thepaw - haemoglobin AAFiltrate pink with some darkred deposit on the paper - haemoglobin AS

Sickle-cell anaemia and the sickle-ceil trait 1 7.27eFiltrete clear yellow (or onlythe palest pink) with adark red deposit on thepaper - haemoglobin SSTake the paper out of the funnel after the first millilitreof filtrate has come through. Do this because oxygenfrom the air oxidizes the insoluble reduced haemoglobinS, and makes it soluble again. When this happens it maystart coming through the paper and make the clear yellowfiltrate from an SS specimen slightly pink.If you are in doubt whether a pale pink filtrate isfrom an SS or AS specimen, leave it until the followingmorning. If any haemoglobin S has got through thepaper and made an SS filtrate pink, it will form a finefeathery precipitate overnight, and leave a perfectly clearsupematant fluid The filtrate from an AS patientmay have a precipitate, but the supematant will stillbe pink.If you still have any doubt about the result of thefiltration test, do this control test. Mix O-9 ml of workingsolution and 1 ml of water. Do the test in the usual way.Both haemoglobin S and haemoglobin A are soluble inthis diluted working solution, so the filtrate using it containsboth these haemoglobins. If the filtrate from the testusing the undiluted buffer is paler than this, some haemoglobinS must have been 1eR behind on the paper, and thepatient must be either AS or SS. He is not AA. This is auseful control to do when seeing if a patient has AS orAA blood.These last two methods are new ones and it has notbeen possible to try them out in many hospitals andclinics before this book was printed. However. theywill probably be a very useful way of telling AS bloodfrom SS blood in areas where this is the commonesthaemoglobinopathy. They may also be useful screeningmethods in larger laboratories where other methods arealso used.Two more things need to be said about these methods.One is that they may not give the correct results inpatients less than about 9 months old. This is becausevery young children may have another kind of haemoglobin(haemoglobin F) in their blood, which may beconfused with haemogtobin S by this method.Also, haemoglobin C behaves like haemoglobin A inthese methods. However, patients with AS blood usuallyhave normal films, provided that there is nothing elsewrong with them, such as iron deficiency. Patients withSC and CC blood have abnormal blood films.If you are in doubt with these methods, you may findit useful to do both of them together and to do the sicklecelltest as well. Both these last two methods were kindlygiven to us by Doctors Huntsman, Barclay, Canning,and Yawson (see references Section 13.33).7.27a Sickle-cell anaemia and the sickle-cell traitThese are important conditions, and it is useful to say alittle more about them.The sickle-celltraitSomeone with the sickle-cell trait is healthy and may beof any age. His blood will sickle and give a pink filtratewith the solubility method. Unless there is something elsewrong with him he will not be anaemic, and he will havea normal blood film.Sickle-cellanaemiaMost patients with sickle-cell anaemia are children, whoare usually very anaemic. Many of them die. Their bloodwill sickle and give a yellow filtrate with the solubilitytest. Their blood films show target cells, polychromasia,and sickle cells. Normoblasts are also often seen.Patients with sickle-cell anaemia suffer from crises. Acrisis is the time of an illness when the patient suddenlygets much more ill and often dies. The more commonkind of crisis is the vascular (blood vessel) occlusive(blocking or closing) crisis in which sickle cells blocksome of the small blood vessels of the body. The childusually has painful bones and joints and may have painful,swollen fingers (dactylitis). He may have pain in thechest and abdomen. He has severe anaemia, fever, andjaundice, and usually has a large spleen. Another andmuch less common kind of crisis is called the aplasticcrisis. The child’s marrow becomes tired of makingblood cells and becomes aplastic or stops working. Thechild thus becomes much more anaemic. The third kindof crisis called the sequestration crisis is seen most commonlyin children between 1 and 3 years old. Many redcells become trapped (sequestered) in the spleen, whichmakes the child very anaemic.Sickle-cell anaemia cannot be cured, but a blood transfusionwill often save a child’s life during an aplastic orsequestration crisis. A good diet also helps these children,and so does folic acid treatment, as it does allhaemolytic anaemias. Sickle-cell crises are often causedby infections. It is thus important to prevent infectionsand to treat them properly when they occur. It is alsomost important that sickle-cell patients should not sufferfrom fever or dehydration, because if they do theiranaemia gets worse. Some clinics give their sickle-cellpatients drugs to prevent malaria and penicillin to preventsome other infections.7.27b ThalassaemiaThis is another group of inherited diseases in which thebody is unable to make haemoglobin in the normal way.The red cells are therefore poorly filled with haemoglobin.and the MCHC is low. The red cells are hypochromic,and there is microcytosis and poikilocytosis.Even though their blood films look very like those of irondeficiency anaemia, they are not cured by iron. In someparts of the world thalassaemia is a common cause ofhypochromic anaemia. One of the easiest ways of distinguishingbetween the hypochromic anaemias of iron deficiencyand thalassaemia is to treat them with iron. If the

7 1 Bloodhypochromia is due to iron deficiency, the anaemia willimprove with iron treatment, but if it is due to thalassaemiairon will do no good. There are other ways offinding out if a patient has thalassaemia, but we cannotdescribe them here.7.28 A simple guide to anaemiaFIGURE 7- 17 will help you to find out why patients areanaemic. When you find that someone’s lips, tongue, andconjunctivae are pale, the first thing to do is to measurehis haemoglobin and if possible, his haematocrit. If hishaemoglobin is more than 10 g % and his haematocritmore than 30%, there is usually no need to treat him. Ifhis haemoglobin and baematocrit are less than thesefigures, work out his MCHC and stain a film byLeishman’s method.If the red cells are hypochromic and the MCHC isless than 32%, look at the stool for hookworm ova andtest it for occult blood. If the patient is a woman, askher about her periods. She may be bleeding too muchfrom her uterus (womb) and be suffering frommenorrhagia.If the red cells are normochromic and the MCHC isabove 32% the patient may have a haemolytic anaemia.Look for polychromasia in the blood film, see if there is areticulocytosis, and look for urobilinogen in the urine. Ifyou find these things the patient has a haemolyticPATIENTS WITH, CLINICAL ANAEMIAanaemia. In many areas, malaria or sickle-cell anaemiawill be the most common causes of such an anzemia, solook for sickle cells and use the solubility method to lookfor haemoglobins A and S. A thick blood film will tellyou if a patient has malaria, if you have not already seenparasites in the thin film.The patient may have a normochromic anaemiabecause he is malnourished (badly fed), or because he hashad an infection for a long time. A few patients will beuraemic. Uraemia often causes anaemia.If many of the cells are larger than normal and arewell filled with haemoglobin, the film is macrocytic. Thepatient probably has a foiic acid deficiency anaemia. Hemay possibly have pernicious anaemia due to vitaminB,, deficiency. If you can, test to see if there is free acidin the stomach. Patients with pernicious anaemia have nofree acid in their stomach. But, if folic acid deficiency iscausing the macrocytic anaemia, free acid will usually befound in the stomach. This is thus a useful test. If patientshave free acid in their stomach they cannot have perniciousanaemia.Very occasionally you will find that leukaemia is thecause of an anaemia.Some anaemias cannot be diagnosed (explained) bythe simple methods that we have described, and someanaemias have more than one cause. Even so, thesemethods will probably be enough to diagnose mostanaemias in most places.RED CELLS FURTHER TESTS DISEASE1HAEMOGLOBINand, orHAEMATOCRITl MACROCYTIC_ _ _ a pernicious anaemia’ folic acid deficiencymalariahaemoglobinless than 10 9%Ehaematocriteless than 30%haemoglobin more than10 9% or haematocritmore than 30%. not worthfurther investigationMCHC mLEISHMAN blood filmthe thickness of the line -shows how many patientsof each kind there arelikely to besickle cell anaemiachronic infectionmalnutrition- uraemiauterine bleedingthe percentage of patients with differentkinds of anaemia often differs greatly fromdistrict to district+HYPOCHROMICstool exam -hookwormsoccult bloo- intestinal bleedingarrows show those diseases where the reticulocytesare increasedill1lllllrII,Fig. 7-l 7 A simple guide to anaemia

THE TOTAL WHITE CELL COUNT7.29 Counting white cellsHealthy people have between 5,000 and 9,000 whitecells in each cubic millimetre (cu mm) of their blood. It isoften useful to know if patients have more or less whitecells than they should have. To find out how many whitecells there are, we take O-05 ml of blood and add it to1 ml of white cell diluting fluid. O-05 ml is jr, ml and isthe same volume that we use for measuring haemoglobin.White cell dilutingAfluid contains acid which will lyse theCounting white cells 1 7.29red cells without harming the white cells. It also containsa few drops of Gram’s stain. This stains the white cells,which would otherwise be colourless, and makes themblue and more easily seen. We add a little blood to amuch larger volume of white cell diluting fluid, so thatwe can make P dilute stained suspension of white cellswithout any red cells in it. By adding O-05 ml (&, ml) ofblood to 1 ml of white cell diluting fluid we have dilutedour blood specimen 20 times (really 21 times, but weforget about this).We put this dilute cell suspension into a glassFROM THE TOP wide middle area, A SINGLE CELLED COUNTiNG CHAMBER\narrow side areasuspension being examinedcAN OBLIQUEVIEWwide middle arearainbow koloured lines)D AN OBLIQUE VIEW A DOUBLE CELLED COUNTING CHAMBERan oblique view meansa view partly from theside and partly .fromon top, specially farge coverglassFig. 7-l 8 Counting chambers

7 1 BloodNeubaucr C0~tbg Chamber (Chamber means a littleroom). This is like a thick microscope slide, but it is verycarefully made and is much more expensive. .4 countingchamber has three special flat areas (places) on it: twonarrower ones on either side and one wider one in themiddle. The middle area and the side areas are separatedby ditches (rivers). The wide middle area is exactly 0.1mm ($ mm) below the areas at the sides. A specialthick, flat coverglass is put across the chant& and restson the two side areas. The coverglass makes a bridgeover the middle area which is exactly 0.1 mm underneathit. There is thus a very carefully made spaceexactly O-1 mm deep between the top of the middlearea of the counting chamber and the bottom of thecoverglass. We put our white cell suspension intothis space.Many very thin lines are drawn in the middle area ofthe counting chamber. These lines cross one another andmake many small squares. These small squares areshown in FIGURE 7- 18 and are called the ruled area. Theruled area can just be seen if the chamber is held up tothe light and looked at very carefully, but it can be seenmuch more easily with a microscope.Some counting chambers have two ruled areasseparated by a ditch as shown in Picture D, FIGURE7-18. These double counting chambers need a speciallylarge coverglass like that shown in Picture G, FIGURE7-23.The ruled area of the Neubauer chamber is shown inFIGURE 7- 19. For the total white count we use the fourcorner blocks each of 16 small squares. These have beenlettered A, B, C, and D in this figure. The very smallsquares in the middle of the cross are marked Z and arefor counting red cells. In our laboratory we do not countred cells. except sometimes in the CSF, and we do notuse these squares. The rulings marked X in this figureare never used.Each of the comer blocks of 16 corner squares isexactly one millimetre (mm) square. So, if the coverglasswas one mm above the ruled area there would be onecubic millimetre (cu mm) above each corner block of 16small squares. But the coverglass is only +s mm abovethe ruled area; so there is therefore only one-tenth of a cumm over each comer block of 16 small squares. Weusually count the white cells in two comer blocks-thatis, two-tenths or one-fifth of a cu mm. To find how manycells there are in 1 cu mm we therefore multiply thenumber of cells we find by five. We also multiply bytwenty, because the blood specimen was diluted bytwenty to make the cell suspension. 5 x 20 = 100; so wecount the cells in two corner blocks and multiply thenumber we find by 100. This is easy: all we do is to addtwo O’s to the number of cells we find in two blocks of 16small squares. For example, if there are 22 cells in blockA and 28 cells in block B, the count is 22 + 28 x 100.This is the same as 50 x 100, or 5,000 cells per cu mm.When you look at the ruled area of a counting chamberyou will see that some of the cells are in the middle ofthe small squares, and some touch the lines at the sides.each of the blocksof 16 small squaresA.B.C. D, is onesquare millimetrecount the 16 small squares in the order shown in block Athat is 1.2.3, ... : you will then follow the ‘snake’ that hasbeen drawn in block Bthree lines very close together+ lmm * lmmTHIS IS ONE OF THESIXTEEN SMALL SQUARESDRAWN MUCH BIGGERcountedcountednotcountedthe small squares marked ‘z’ are used for counting thered blood cells : this method is not described in thisbook : the parts marked ‘x’ are not used at allI mmmmin any one squarecount all the cellstouching the topand right handlines : leave outthe cells touchingthe bottom andleft hand linesFig. 7-19 The ruled area on a Neubauer countingchamberWhen you count the cells in a square, it is a useful rule tocount only those cells which touch the top and right-handlines round a square, and not to count the cells whichtouch the lines at the bottom and left-hand edges qf asquare. If a cell touches the left-hand edge, it will becounted with the cells in the square next to the left. If acell touches the line at the bottom of a square, it will becounted with the cells in the square below.When doing a white cell count the important thing tolearn is the right way to put a coverglass on to a countingchamber. The coverglass and the narrow side areas musttouch one another closely, and there must be no dustbetween them. Only if they do this, will there by exactly0~ 1 mm between the coverglass and the middle area.Follow the instructions for putting on the coverglass onthe chamber very carefully. Remember especially neverto press on the coverglass except over the narrow sideareas. If you press on the coverglass in the middle, it willbreak.

Counting white cells 1 7.29THESE ARE BOTH VIEWS OF THE RULEDAREA LOOKING FROM THE TOP AND THE SIDEIf the coverglass was 1 mm above theruled area there would be 1 cu mm ontop of each of the corner blocks of 16small squares, but the coverglass is onlyl/10 mm above the ruled area, so thereis only l/10 cu mm above each of thecorner blocks of 16 small squareslmn..?,‘*..!:?nr.‘::.-1, .’ ..~~~~~r~~mm..,. 1.EXAMPLE,If there were 22 cells in block A and 36 in block 6 the answerwould be:-small squares each of which is l/10cu mm. These together make l/5 cu mm.Multiply the number of cells you find byfive. Multiply it also by 20, because theblood has been diluted twenty times withdiluting fluid. 5 x 20 is 100, so multiply thenumber of cells you find in two cornerblocks of 16 small squares by 100.Fig. 7-20 The volume of a Neubauer counting chamberMETHODTHE TOTAL WHITE CELL COUNT, FIGURE 7-211. Make sure that both the coverglass and the countingchamber are really clean. If necessary wash themwith soap and water and dry them with a soft cloth.Put the counting chamber on the bench. Put thecoverglass on top of it. Put your index finger on top ofthe coverglass. Very gently, without pressing, move thecoverglass round and round. This will make any dustbetween the coverglass and the side areas fall into theditches. The coverglass and the side areas will thentouch one another closely and leave exactly 0.1 mmbetween the coverglass and the ruled area.2. Put the coverglass towards one side of the chamber.Put one of your thumbs on each edge of the coverglass.Press gently over the narrow side areas and, whilestill pressing, push the coverglass into the middle of thecounting chamber, as shown in Picture A at the top ofFigure 7-23. As you have read, s/ways press over thenarrow side areas as in Pictures 2 and 3. Never press onthe middle of a coverglass. If you do the coverglass willbreak, as in Picture 4.When the coverglass is properly in place you will seethe coloured lines of the rainbow or spectrum betweenthe narrow side areas and the underneath of the coverglass.These are shown in Picture A, Figure 7-23.These coloured lines mean that the coverglass andthe narrow side areas are touching each other closely.You will find, when you pick up the counting chamber,that the coverglass will have stuck to it. On some countingchambers the narrow side areas are rough (ground).so you will not see rainbow colours, and the coverglasswill not stick to them.5. Measure 1 ml of white blood cell diluting fluid intoa bijou bottle. You may find it useful to keep a l-mlpipette in the cork of the stock bottle of diluting fluid.6. Fill the blood pipette to the 0.05-ml mark. Takeblood from the ear or finger or from a venous specimen.Make sure you mix the specimen bottle well before takingthe sample. Fill your blood pipette as described inFigure 7-l.

put the coverglass at ageedge of the chamber andgently move it inro2 the middle : presscarefully on then \ sides as you/ ,-\ x6zthere is 1 ml of white celldiluting fluid in this bottle3this blood pipett e has beenfilledto the ’ U.05 - -- ml markdcoverglassII / 1counting chamber’ L only Press on the sides of thecoverglass over the narrowside areas!I4 rA Al

What an abnormal total white cell count means 1 7.367. Wipe the outside of the pipette clean and blow theblood into the l-ml bottle of white cell diluting fluid.Rinse the blood pipette once by drawing up the fluid tojust above the 0.05ml mark and blowing it out again.8. Put the cap on the bottle and shake it gently.9. Take a clean Pasteur pipette and draw the whitecell suspension in and out of it once or twice.10. Using the pipette, fill the space between thecoverglass and the middle part of the counting chamberwith the white cell suspension. Fill the chamber in onesteedy movement- There must be no bubbles and nosuspension in the ditches. You will find that the suspensionwill flow into the chamber on its own.Turn to Figure 7-22.1. Move the condenser down. Look for the ruled areawith the low power objective.2. If you cannot find the ruled area, find the edge ofthe ditches.3. Focus upwards until you see the top of the ditchand the surface of the wide middle area.4. Move across the wide middle area until you find theruled area.Count the cells in two corner blocks of the 18 smallsquares. When counting a corner block move throughthe 16 small squares following a snake-lie path drawnin block 8 in Figure 7-19. Add together the number ofcells found in each block. Add on two 0’s. The figure youget will be the number of white cells in 1 cu mm.Picture A, FIGURE 7-23, shows you what a well-filledcounting chamber should look like. The coverglass is inthe middle. You will see the coloured lines of the spectrumover both side areas; there are no bubbles in thesuspension; and no suspension in the ditches. All theother pictures show badly filled counting chambers. InPicture D the coverglass is at one edge of the countingchamber. In Picture B the coverglass is not straight andthere are bubbles in the suspension. In Picture E suspensionhas into the ditches. In Picture C a coverglassthat was m % t for a single celled chamber has been puton a double chamber. You will see that it is too small andthat the ruled areas are not completely covered. Theruled areas must be well inside the edge of the coverglass.You will see that the double coverglass in Picture G ismuch bigger than the single coverglass in Picture F.AGOODre coverglass ie themiddle of the chamber rainbow coloursBADherecBthe covergl>ss is crooked andthere are bubbles in the suspensionBADthis is a coverglass for a single cell chamberused on a double cell chamberBADD-‘the coverglass is at the edgeof the chamber7.30 What an abnormal total white cell countmeansBy an abnormal white count we mean one with morethan 9,000 or less than 5,000 white cells per cu mm.When a patient has too many white cells (leucocytes) hehas a leucocytosis. When he has too few white cells hehas a leucopenia (penia means few). Many infections withbacteria produce a leucocytosis. Very rarely you willfind a patient with a white count of 100,000 or evenmore. This may mean that the patient has a very baddisease of his blood called leukaemia.for singlecell chamberfor doublecell chamber, IElF 6Fig. 7-23 Well and badly filled counting chambers

,.,L I /p%‘,.,‘__^_,: 7 1: B!podLeucopenia is much less common than leucocytosis.Typhoid fever is an important cause of a moderate leucopenia,say, 4,000 cells per cu mm. Sometimes there maybe very few white cells indeed because the marrow isdiseased. As you have read, a diseased marrow whichproduces very few cells or none at all is said to beaplastic.Nothing more than this can be said here about themeaning of an abnormal total white cell count. Btprepared therefore to&d a few patients with very highcounts and a few with very low counts. If you find anoccasional very unusual count, your method is probablyright, and the patient’s white cell count is probably veryabnormal.Total white cell counts will seldom be needed in healthcentres. But they will often be wanted in hospitals to helptid the cause of a fever or, less often, the cause of ananaemia.FIELD’S STAIN FOR THICK BLOOD FILMS7.31 Why a thick film is so usefulField’s stain was first made to show malaria parasites inthe blood. but it will also show trypanosomes, microfilaria.and Borrelia. Because these can all be seen in a thinblood film, you may wonder why we need thick bloodfilms. This is the reason. In a thin film the blood is spreadout so thin that, unless there are very many parasites intbe blood, we are unlikely to find them. This is becausewe can only look at a very small volume of blood withan oil immersion objective, unless of course we searchfor a very long time indeed. In a thick film we cansearch a much larger volume of blood and have a betterchance of finding parasites when there are not verymany there.Leishman’s stain contains methyl alcohol which fixesthe blood cells. You will remember that by fixing wemean that the methyl alcohol kills the cells and keepsthem looking the same as when they were alive. Field’sstain is made with water and contains no methyl alcohol.So, when a thick film of blood is put into Field’s stain,the water in the stain lyses (breaks open) the red cells andwashes the haemoglobin out of them. But the malariaparasites inside the cells are left behind when the haemoglobinis washed away and are stained, so also are thewhite cells. If all the haemoglobin is washed away the redcell membranes (coverings) are also stained, and the fieldof view in the finished film will be full of purple debris(rubbish). This makes it difficult to see the parasites. But,of the haemoglobin is left in thefilm. you will seeiSsomeparasites and white cells stained blue and purple againsta CLEAR orange-brown background, which is theremaining haemoglobin. This clear background makes iteasy to see the parasites and is explained in FIGURET-24.Thick films are not easy to make. If the film is stainedbadly too much haemoglobin may be washed away, andthe film may even be washed off the slide completely.Only with care and practice is it possible to make a goodthick film every time. But, when a good film is made, it isbeautiful, and it gives a sure diagnosis easily and rapidly.The main points to watch are the thickness of the film,how dry it is, how long it is stained for, and how much itis moved about in the jars of stain and water.METHODFIELD’S STAIN, FIGURES 7-25 AND 7-25THESTAINKeep Field’s sta.in A and Field’s stain 8 in two jarswith caps that are wide enough for a slide to go throughthem-150-ml screw-capped jars work very well. Jarsof this kind are listed as ML 62. Keep them very nearlyfull; otherwise it will be difficult to beach the stain withthe film. Put the caps b&k on the jars when they are notbeing used.MAKING THE FILMThis is very impofiant. The film should be about thesize and shape of a postage stamp-say, 2 cm x 14 cm.It should be thick enough so that the writing in a newspaperor the hands of a watch can only just be seenthrough it. Use the corner of another slide to spread outthe blood. A common mistake is to make thick films toothin. Thick films must be thick! They are usually made inthe wards; so, show the ward staff, and particularly thestudent nurses, how to make good ones. If very badfilms are sent to the laboratory, don’t look at them,because it will be impossible tcb give a true answer. Askfor another film, or, better still, go to the ward and makeanother thick film yourself. Show the person who madethe bad film how to make a good one. One way of teachingthe ward staff is to stick films, good and bad, to acard, to label them and to leave them on the ward noticeboard as examples.The film must be dry enough. A few minutes in awarm place, such as on top of the microscope lamp, willmake the film dry enough to stain. Films that are too drydo not stain well. You will also find that fresh films stainbetter than old ones. Use a big enough drop of blood andbe sure that the film is flat (horizontal) while it is drying.STAINING1. Dip the slide in the blue stain A for one or twoseconds, moving it about gently. Let it drain for amoment, by touching the bottom of the slide on the topof the jar.2. Wave the slide gent& in a cup of clean water. 2-3seconds is usually enough, by which time the stain willusually have stopped flowing from the film.3. Dip the slide for one second into stain B. Let it drainfor a moment by touching the bottom of the slide on thetop of the jar.4. Wave the slide gently for 2-3 seconds in clear

I1.‘.)?6),.I~_I/” ,1,~ Why a thick film is so useful 1 7.31these are red cellslooked at from the sideA THIN FILM the haemoglobin of a thin film isnot washed away when it is stained, this is a white celithis is a slide lookedat from the side\ this is a red cell with a \malaria parasite inside itnucleus of white cellPlate 18 etcA THICK FILMunstainedin Field’s method of staininga thick blood film we try toleave some haemoglobin inthe film : in Giemsa’s method ofstaining a thick film we wash itall away : Giemsa’s method is notdescribed hereGQQD TIthis is a heap of red cells, some ofthem with malaria parasites insidethem and some of them withoutstainedno remaininghaemoglobinnucleus of white cellif the film is well stained theparasites are left in a clearlayer of haemoglobinFig. 7-24Thick and thin blood filmsif the film is overstained and allthe haemoglobin is washed away,much debris is also stained andthe parasites are hard to seeslideA GOOD THICK FILM IS THE SIZE OF A POSTAGESTAMP AND SO THICK THAT YOU CAN JUST SEE THEHANDS OF A WATCH OR NEWSPRINT THROUGH ItFig. 7-25A good thick filmjg;\?gruse a big enough dropof blood and make surethat the film is flat(horizontal) while itdries

7 1 BloodTHE SLIDE MUST ONLY BE IN THE STAINSAND THE WATER FOR A VERY SHORT TIMEthis is the jar ofField’s stain ALthis is the cap of the jar. it willba put back on the bottle as soon asstaining is finished to stop the &indrying up : the cap must also be putback on the jar of Field’s stain 6-Fig. 7-?6_ ,_ ,full of stain, the film can easily’reach the stain in the iarField’s stainwater again. While the film is in the water and thestains, most, but not all, of the haemoglobin shouldwash away, and the film should become much clearer.5. As soon as the film is stained leave it upright in arack to dry, or dry it with a hair drier (see below).When the film is quite dry smear the whole film withoil. Using the low power objective look all over the filmfor the best place to look at with the oil immersionobjective. The place to look should be a clear orangebrowncolour and will probably be thick enough to havecracked as the film dried.There are many important details. The first is that thestaining and washing times are very short indeed. Dr.Field himself said that the film should be one second inboth stains A and B and one second in the water for thefirst time. He said it should be 2-3 seconds in the waterthe second time. The film must not be so long in eitherthe water or the stains that all the haemoglobin washesout. Too long in the stains and water will stain the redcell membranes, and the field will be full of purple debrisagainst which it will be hard to see the parasites. Jars ofstain differ in the times that films need to give a reallygood result. Very dry old films need a longer time forboth staining and washing than do moist (not quite dry)fresh ones. When you have made a new batch of stain trystaining a film for the times given above. Lengthen thesetimes if you do not make a good film the first time. Filterthe stains when they are first made and again when ascum forms on the surface.A good way of drying films rapidly is to use a domestic(for the home) hair drier. A hair drier is much betterfor drying films than a Bunsen burner. It blows hotair and dries films almost immediately. A hair dryermakes it possible for someone to bring a thick filminto the laboratory and to stain it, dry it, and lookat it all in about 5 minutes. He need waste no timewaiting.If the film is well made, white cells are easily seen, andit is sometimes possible to tell the various kinds of whitecell-neutrophils, eosinophils, and lymphocytes. Thesewhite cells and any parasites there may be in the filmshould be seen against an almost empty clear orangebrownbackground. Platelets also stain as small bluishobjects with a pink centre.7.32 MalariaMalaria is caused by four species of parasitic protozoabelonging to the genus (tribe) Plasmodium. These plasmodiaspend part of their life in man and part of their lifein certain kinds of mosquito. In man the plasmodia(malaria parasites) live and grow inside the red bloodcells and destroy them. Plasmodium falciparum causesmalignant tertian or MT malaria. Plasmodium vivaxcauses benign tertian or BT malaria. Benign (not serious)tertian (three day) and malignant (very serious, killing)tertian are old names for malaria fever, but they are stillused. Malaria is also caused by Plasmodium ovale andPlasmodium malariae. The word Plasmodium is oftenshortened to ‘P.‘; so we have P. vivax, P. falciparum, etc.Some species are common in some countries and somespecies in other countries. In some places only onespecies of parasite is seen; in other places two or threespecies are seen, and in a few places all four species arefound.Not only are there four species of malaria parasite, buteach species grows through several stages inside the redblood cells. Each stage of growth has a special name,such as merozoite, trophozoite, schizont, or gametocyte.Each stage of each of the four species differs a little fromthe others.

Ilymphocyteb 0 ayoung trophozoites ofP. falciparum - ‘&lT rings’ a&I11 )$adouble chromatingdotRII*&schizontIplatelet84lymphocyteThese are all oil immersionthis gametocyte has roundedends and diffuse chromatinc trophozoites of P. falciparu‘in, d’these have grown slightlv largerthan those shown in-picture %views.l[c kor r eZutt oni ’ 46@&:;; Q$ (1e\Q *D gametocyte of PyfalciparumFig. 7-27 Some thick films stained with Field’s stainthe species is not certain becausethe tip of the tail is hidden behindpart of the body., maturingyoung trophozoite‘MT ring’remains of red ccl IFig. 7-28 Plasmodium falciparum in the blood

FIGURE 7-28 shows the stages through which P. falci-Parum grows. The smallest and youngest form is themerozoite, which is shown in Picture A. This then goesinto a normal red cell (Picture B) and grows to become atrophozoite (Picture C). The trophozoite of P. fafci-Parum is like one of the rings that people wear on theirfingers. It is CirculW with a stone at the edge of the circle.‘he stone in the ring is called the chromatin dot andStains a deep reddish purple with Field’s stain; the rest ofthe ring stains a pale blue. These rings are very small andare about the same size as platelets. It is easy to distinguislha young trophozoite of P. falciparum from a plate!et,because a trophozoite has a ring and a chromatin dot,while a platelet has neither.As the parasite matures (grows) it passes through thestages shown in Pictures D and E. The trophozoite getsbigger, and the vacuole (hole) inside the ring disappears.The chromatin dot grows larger and then breaks up intoseveral pieces as shown in Picture F. A parasite that isabout to break is called a schizont. Each piece of theschizont has a little ring of blue cytoplasm around it.When the schizont breaks, the red cell also breaks(Picture G). These broken pieces of the schizont becomethe merozoites which infect several new red cells. This isone way in which plasmodia multiply (become many). It ispart of what is called the life cycle (life circle) of the pafasite.The life cycle is completed in the mosquito whichtransmits (takes) maleria from one person to another.Some of the merozoites don’t become schizonts butgrow into gametocytes. One of these gametocytes isshown in Picture H. Some of these gametocytes are male(like a man) and some female (like a woman). Inside themosquito they mate (marry). The gametocytes of P. faici-Parum are shaped like a new moon-‘crescent shaped’-but the gametocytes of the other species are round. MTcrescents are therefore very easy to recognize. Three ofthem are shown in Picture D, FIGURE 7-27.MT malaria caused by P. falciparum is a common andserious kind of malaria. One of the most common reportson a thick film is therefore MT rings + + +. This meansthat there are many young trophozoites of P. fakiparumin the film. YOU can see these in Pictures A and B inFIGURE 7-27. In Picture B the trophozoites have growna little larger than those shown in Picture A.7.33 A diagram of the human plasmodia (FIGURE7-29)When you look at a blood film it is always very importantto make quite sure when there are malaria parasitesin it and when there are none. Often, it is also importantto tell which species of Plasmodium a patient has,because the treatment for the various species ofPlasmodium is different. Chloroquine and drugs like itkill all four species of Plasmodium when they are in thered cells. They also cure a patient’s fever. But, after anattack of fever, P. vivax, P. malariae, and P. ovale maystay in the tissues where they are not killed by chloro-Wine. P. falciparum does not go into the tissues after anattack of fever; so chloroquine kills all the P. falciparumparasites in the patient’s body.If the parasites stay in the tissues and are not killed,they may go back into the red cells later. If they do this,the patient will relapse. That is, he will get another attackof malaria fever some time later.When patients have malaria caused by P. vivax, P.ovale, and P. malariae they should thus be given chloroquine.They should also be given primaquine for 14 daysto kill the parasites in their tissues. The rule is this:P. falciparum malaria : chloroquine alone isenough.P. vivax, P. malariae,P. ovale malaria : give chloroquine andprimaquine.In some countries there are so many infected mosquitoesaround that a patient is more likely to get anotherattack of malaria from another mosquito bite than he isfrom the parasites remaining in his own tissues. In thesecountries patients are often not given primaquine, eventhough P. vivax. P. malariae, or P. ovale is causing theirmalaria.In countries which are trying to drive out or eradicatemalaria, it is especially important to find out whichspecies of Plasmodium a patient has, so that he can begiven the right treatment to kill all the parasites in hisbody. If every patient with malaria can be properlytreated, malaria can be driven out of a country.FIGURE 7-29 will help you to recognize the fourspecies of Plasmodium. Row A shows the young trophozoitesof each species. Row B shows the mature trophozoites.Row C shows the plasmodia as they are about todivide-the schizonts. Row D shows the male malariaparasites-the male gametocytes. Row E shows thefemale gametocytes. The first column is P. vivax, thesecond is P. malariae, the third is P. falciparum, and thefourth is P. ovale. Here are some of the more importantthings to. look for with each species.P. vivax is the only species which makes the red cell getbigger and still keeps it round (Pictures 1. 5, 9, 13, 17).P. ovale makes the red cell bigger and also oftenmakes its edge irregular (rough). The red cell of P. ovaleis often oval (egg-shaped) like its name-otjaale (Pictures4. 8, 12, 16, 20).Only P. ovale and P. vivax make round red dots(spots) on the red cell (Pictures 5, 9, 13, 17 and 4, 8, 12,16, 20). These dots are called Schuffner’s dots. Schuffner’sdots are more often seen with younger (smaller)trophozoites with P. ovale (Picture 4) than with P. vivax(Picture 5).Multiple infections (more than one trophozoite in ared cell) are quite often seen with P. vivax (Picture 1).They are very often seen with P. falciparurn (Picture 3).As you have read, all malarial trophozoites have adark purple dot called the chromatin dot. This dot isusually large in P. malariae (Pictures 2, 6). In P. falciparumthe chromatin dot is small and is often double(there are two dots side by side, Pictures 3. 7).

P.vivax P.malariae P.falciparum P.ovaleyoung trophozoitescommondotPlates 26 & 27Plates 34 & 35growing trophozoites ’Plate 42irregularSchuff ner’s dotsCPlates 20 & 21 with irregularvacuoles (holes)schizontsthese are notoften seen in the,/peripheral bloodred cell often has anoval shape - ‘ova@Plates 22 & 23male gametocybs n,the chromatin is diffuse in all male gametocytesirregular,’red cells. coarse tenchromatinIfemale gametocyb?s Schuffner’s dots &wious (the chromatin in all female gametocytes :is in a lump at the edge of the cell \.Fig. 7-29 A diagram of human plasmodia

.!’,” i _l&&i -, ” _!“i’7 1 Blood‘yAll species make tiny granules of a blackish materialcalled malarial pigment. P. malariae makes more pigment,makes it earlier, and keeps it in larger lumps(Pictures 6, 10, 14) than do the other three species.The chromatin of the male gametocyte breaks intopieces in the cytoplasm (Pictures 13, 14, 15, 16). Thechromatin of the female gametocyte is in one lump,which is usually at one side of the cell (Pictures 17, 18,19,20).The gametocytes of P. faZc@arum (Pictures 15, 19)are shaped like a new moon (crescent shaped) and arethus very different from the round gametocytes of otherspecies. It is not important to be able to tell a malegarnetocyte from a female one.Diagnosing the species of malarial parasite from athick film is usually easy, but it can be dimcult,especially when there are only young forms in the film.Look carefully at several parasites. Sometimes a patienthas more than one species of Plasmodium in his blood,so be careful. It is usually easier to tell the species ofmalaria parasite in a thin film than in a thick one.If you think that a patient has malaria, but you cannotfind any malaria parasites in a blood film, look againsome hours later. All the parasites may be at the merozoiteor early (young) trophozoite stage and may be hardto find. Some hours later, when they have grown, theymay be easier to see. One negative thick firm does notmean that the patient has not got malaria.7.34 The meaning of a positive thick film in malariaWherever there are many trophozoites in a blood film,the patient is ill and needs treatment. The drug that he isusually given is chloroquine. Chloroquine is quite expensive;so patients who are not very ill are sometimes givenmepacrine, which is cheaper, but not quite as good insevere cases.There is a very serious kind of malaria called cerebralmalaria (malaria of the brain) which is always caused byP. Jalciparum. The patient usually has a high fever. Heoften has fits and usually loses consciousness (he seemsto be asleep but cannot be woken up). These patientsmust be given an injection of chloroquine or quinine orthey will die. It is thus very important to be able todiagnose cerebral malaria quickly and certainly. This isone of the most important uses of Field’s stain.Blood cells infected by P. falciparum become ‘sticky’and stick to the inside of the capillaries and block them.It is this blocking of the capillaries of the brain thatcauses the signs and symptoms of cerebral malaria. Theschizonts of P. Jvlcipantm (Picture 11, FIGURE 7-29)are very seldom seen in a blood film-they are stuck tothe inside of the blood vessels. But, when the schizonts ofP. farcipanrm are seen in a blood film. they mean that thepatient has a very bad attack of malaria. Schizonts in ablood film usually mean that a patient has cerebralmalaria.People who live in countries where malaria is commonmay have a few malaria parasites in their blood and seemto be healthy. For example, you may see a gametocyte ofP. falciparum (a crescent) in the blood of someone, particularlya child, who has no fever and looks healthy.These patients are fighting the malaria parasites quitewell, and they do not usually need any treatment. But themalaria parasites may start to win at any time (they maystart multiplying). When they do the patient will havefever and needs treatment.A patient may also have malaria and another disease,pneumonia for example. If you find malaria parasites thisdoes not therefore always mean that they are the mostimportant cause of a patient’s illness.Malariain the placentaMalaria parasites sometimes infect the placenta or ‘afterbirthand harm it so that a baby is born smaller than heshould be. This can easily be prevented by giving allmothers malaria tablets while they are pregnant. Thiswill only be necessary if there is much malaria in adistrict and many new-born children are underweightbecause their placentae are infected. The best way tofind out is to examine the placentae from a group ofmothers, especially those who have given birth to smallbabies.METHODEXAMINING THE PLACENTA FOR MALARIA PARASITESFind the rough or mother’s side of the placenta whichjoins on to the inside of the womb. Cut off a small piecewith a pair of scissors. Hold it in forceps and ‘dab’(touch) it on to a clean slide. Be careful only to touch theslide once or the film will be too thick. One touch with apiece of placenta will leave enough cells to stain andwill not distort (spoil) them.Stain and examine your film bv Field’s method.You will find malaria parasites, as you would in a bloodfilm.7.35 Relapsing feverRelapsing fever is much less common than malaria, butin places where it is seen it is likely to be one of the mostimportant reasons for looking at a thick blood film. Themicro-organism that causes relapsing fever is a long,thin, curved bacterium called Borrelia duttoni. It lookslike a snake and has been drawn in Picture C, FIGURE 7-27. Relapsing fever is called relapsing fever because thepatient has fever for about a week and then gets better fora few days, and then after this he gets worse again or‘relapses’. During the days when the patient feels well theBorrelia disappear from the blood and cannot be foundin a thick film. It is especially important therefore tomake the films when the patient has a fever. If you do notfind Borrelia the first time you look, make several filmson several days when the patient has fever. Borrelia canalso be found in a wet blood film.

Sleeping sickness or trypanosomiasis 1 7.367.36 Sleeping sickness or trypanosomiasisThese are two names for the same disease. Trypanosomesare flagellates (protozoa with flagella) whichswim actively in the blood. A trypanosome has beendrawn very large in FIGURE 7-30, as you can see fromthe size of the red cell. A trypanosome has a long tail orllagellom and a long fln like a fish down one side (a lin isthe ‘arm’ of a fish). This Gn is called the undulatingmembrane because it goes up and down or undulates.FIGURE 7-30 shows the undulating membrane and alsowhat a trypanosome would look like if it were cut in half.In a living, active trypanosome the undulating membranegambiense. But they both look exactly the same in theblood. We therefore report a positive fihn as‘trypansosomes present’ and use the plus notation (seeSection 4.4) to say how many there are.If trypansosomes are found in a patient’s blood heneeds immediate treatment in hospital for several weeks.Suramin and melarsoprol are two of the drugs that hemight be given.7.37 FilariasisSeveral kinds of worm live in the tissues. They live in theskin, in the veins, or in the lymph vessels, and not in theif the trypanosomewere cut in halfhere is part of a red cellwith which to compare thesize of the trypanosomeFig. 7-30 A trypanosome in the bloodmoves too fast to be seen. But, as a trypanosome dies, itsundulating membrane moves more slowly and can besee* quite easily with an oil immersion objective.Because trypansosomes move so actively when theyare alive, it is usually easier to find them moving andunstained in fresh blood, than it is to find them dead andstained in a thick film. This is because something whichis moving is easier to find than something which is stillitis much easier to see a moving animal in the bush thanit is to see a still one. Because we so easily see somethingmoving we can search for trypanosomes with a highpower objective instead of au oil immersion objective. Inthis way we can search more blood for trypanosomesand are more likely to find them than when there are onlya few in the blood. Trypanosomes can be found by lookingfor something moving in a drop of fresh blood undera coverslip, but it is better to use the concentrationmethod described in Section 7.38. This method concentrates(gathers together) the trypanosomes so that theyare more likely to be found. The concentration methodcombines the advantages of both concentration and thesearch for movement.In Africa two species of trypanosomes cause diseasein man: Trypanosoma rhodesiense and Trypanosomagut like the hookworm. The most important group ofworms which live in the tissues are called filariae. Theycause a disease called filariasis and produce youngworms called microfilariae (little filaria). Most of thesemicrofilariae can be found in the blood, but the microfilariaeof 0. volvulus are an exception. These microlilariaeare found in the skin and are described in Section11.14. Blood microfilariae are easily found in a bloodfilm, or with a concentration method.Part of a microfilaria is drawn in Picture E, FIGURE7- 11, and a complete microfilaria is drawn in Picture F,FIGURE 7-27. As you will see, microfiliariae are muchlarger than plasmodia. Borreliu, or trypanosomes.Microfilariae have a head, a body, and a tail. Thesejoin on to one another, as in a snake, and it is difficult totell where the tail begins and the body ends. Some microtilariaealso have a sheath (loose outer coat). This islike the dead skin of a snake. You will see that themicrofilaria in Picture F, FIGURE 7-27, has a sheathwhich is longer than the worm itself and that this sheathsticks out beyond its head and its tail.At least five species of microfilaria can be found in theblood, but in any one place you are unlikely to have todistinguish more than two or three species. In Uganda,

7 1 WoodThree harmful species - sheathed\AA ~rrt-uwnriahnnmmftiusually much more common in night blood - nocturnal periodicityno nuclei in the tip’of the tailmore common in night blood - nocturnal periodicityLoa loamore common in day blood - diurnal periodicityzythis is a red cell ‘$7-5 Pm drawn to the same\scale as the microfilariaethe black dots are nucleilate 53IV nucletie tallTWO harmless species -unsheathedno sheathDipetalonema& periodicity variablea-perstanshnuclei right to the end ,TMansonella ozrardiequally easily seen by day or night - non-periodicFig. 7-31 Microfilariae in the bloodend of the tail

A concentration method ! 7.38for example, only two species are found--D. perstans iscommon and L. Zoa is rare. Find out which species arefound in your country and learn to recognize them.When you are trying to find the species of a microfilaria,first see if it has a sheath. If a microfilaria has asheath, it is either W. bancrofti, B. malaJ$, or L. loa. If ithas not got a sheath, it may be either D. perstans or M.ozzardi. It may also be a normally sheathed microfilariawhich has temporarily lost its sheath and is growing anew one. Next look at the end of the tail because eachspecies of micromaria has a slightly different tail. Seehow far the nuclei go towards the end of the tail and howthey are placed at the end of the tail. The five species ofmicrofilariae commonly found in the blood have beendrawn in FIGURE 7-3 1. The sheaths and the differenttails of each species are well shown.All the species of worm which produce sheathed microfilariaecause disease in man. The two which producemicrofilariae without sheaths are closely related to oneanother and cause no disease. If therefore you see amictofilaria with a sheath it must be harmful. If,however, you see a microfilaria without a sheath it maybe harmless or it may be harmful, because it may be aharmful microfilaria which has lost its sheath and isabout to grow a new one. Look carefully at the tail andtry to base your diagnosis of the species on several microfilariae.Perhaps you will find one with a sheath. In mostparts of the world it is uncommon for a patient to havemore than one species of microfilaria in his blood.W. bancrofti and B. malayi are close relatives andboth have sheathed microfilariae. They are closelyrelated to one another and cause the same kind ofdisease. The adult worms of these two species live in thelymph vessels and block them. The lymph vessels arevery small, thin tubes which take a watery fluid calledlymph from the tissues back into the blood stream. Whenthe lymph vessels are blocked the lymph stays in thetissues and the tissues swell up (get bigger). A patient’slegs may swell until they look like the legs of an elephant.This gives this kind of filariasis its name--elephantiasis.The third species of adult worm which has sheathedmicrofilariae is L. loa. This worm causes soft, warm,short-lasting swellings on the skin which are not usuallyharmful.Because the unsheathed microfilaria, D. perstans andM. ozzardi are quite harmless, the patient in whom theyare found needs no treatment. But, if any of the sheathedmicrofilariae, such as W. bancrofti, B. malaj$, or L. loaare found, the patient should be sent to hospital andtreated. He will probably be given tablets of a drug calledhet razan.Some microfilariae are more common at special timesof the day or night, and are said to show peridicity. W.bancrofti is usually much more common at night and issaid to show nocturnal (night) periodicity. B. malayi isalso more common at night. L. loa is mostly seen byday-diurnal or daily periodicity. In some people and insome places A. perstans shows diurnal periodicity, and inothers it shows nocturnal periodicity. M. ozzardi may befound equally easily at any time and is said to be nonperiodic.When you are looking for particular microfilariae,take blood at the best time to catch them.When microfilariae are alive they move about in theblood like very active snakes. It is best to look for themmoving about in fresh blood in the same way as trypanosomes.The next section tells you how.7.38 A concentration methodBlood is taken from the patient and put into a solution tostop it clotting (an anticoagulant solution). The blood isthen centrifuged. This separates the blood into a layer ofplasma on top and a layer df red cells at the bottom. Inthe middle there is a thin, dirty white layer of white cells.Some of this white cell layer is removed with a Pasteurpipette and looked at under a microscope. You are morelikely to find microfilariae or trypanosomes in this layerthan you are to find them in plain blood. The parasiteswill have been concentrated or gathered together into onepart of the blood. This is what we mean by a concentrationmethod.METHODCONCENTRATING MICROFILARIAE AND TRYPANOSOMES,FIGURE 7-32USE Fk?SH BLOOD AND DON7 WASTE TIME1. Take about 3-5 ml of blood and add it to 0.5 ml ofsodium citrate to stop it clotting. This sodium citratesolution has been made for the Westergren ESR-lookat Section 3.42a. You can also use solid sequestrene asan anticoagulant, but a liquid anticoagulant solution isbetter.2. Centrifuge the blood for 10 minutes. If you arelooking for trypanosomes, centrifuge it as fast as youcan. If you are looking for microfilariae centrifuge theblood at about 1,000 revolutions (turns) a minute. Onethousand revolutions a minute is about as fast as youcan turn a hand centrifuge, or about a third as fast as anelectric centrifuge will go. At full speed most ordinaryelectric centrifuges turn at about 3,000 revolutions aminute.3. After centrifuging you will see three layers-a layerof plasma, a layer of white cells, and a layer of red cells.4. Using a Pasteur pipette take off the supernatantplasma until you get to the white cell layer.5. Take as much of the white cell layer as you can andput it on a slide under a coverslip. If you are looking formicrofilariae take some of the red cell layer also and putthis under the coverslip too. Some people like to sealthe coverslip by the method showh in Figure 7-14.6. Look all over the coverslip by the methad shown inFigure 6-14. Don’t use too much light-see Section6.15. Use a low power objective for microfilariae and ahigh power objective for trypanosomes. Look forsomething moving about very actively like a fish. If yousee something moving around like this which is about as

7 1 Bloodtake some of thewhite cell layerplasma and throwblood in anyanticoagulantUGEI microfilariae ortrypanosomes willbe in this layerLOOK FOR SOMETHING MOVING, use thelow power objective for microfilariaeand the high power objective whenlooking for trypanosomesTHIS IS A METHOD FORFINDING MICROFILARIAEAND TRYPANOSOMESFig. 7-32 A concent:=?ion te”* J, &-- IUI IluLrofiiariae -’ -.and trypanosomeslong as two red cells, it must be a trypanosome. If it ismoving about in the same way but it is as long as, say,twenty or thirty red cells, it must be a microfilaria.You will not be able to tell one species of microfilariafrom another while they are moving about in wet blood.If you want to find which species a microfilaria belongsto, you will have to stain a blood film, preferably a thinblood film. Search the whole film with a low powerobjective.Blood must be looked at quickh, besore the trypanosomesdie. Make sure that blood is taken straight fromthe ward to the laboratory. no the concentration methodquickly. You should be searching the film before theblood has been taken from the patient 20 minutes.7.39 The ESRIf blood is taken from someone and prevented from clotting,the red cells will slowly fall and leave clear plasmaon top of them (see FIGURE l-4). The rate (speed) atwhich the erythrocytes (red cells) sediment (fall) is calledthe ‘Erythrocyte Sedimentation Rate’ or ESR. It is alsosometimes called the blood sedimentation rate or BSR. Itis the distance in millimetres that the red cells fall in onehour.Blood must be taken into a special anticoagulantsolution. and the cells must be allowed to fall in a specialtube (ML4). There are several ways of measuringthe ESR. We shall describe the Westergren ESR, whichuses 3.8% citrate solution and the Westergren tube.The tube of blood is put into a special Westergren rack(ML II).The red cells of a healthy man fall less than 10 mm inone hour in a Westergren tube. The red cells of a healthywoman always fall less than 14 mm in one hour, unlessshe is pregnant. But in many diseases a patient’s red cellsfall faster than this. The fall is greater in some diseasesthan it is in others. For example. the ESR is very high inleishmaniasis and trypanosomiasis and is normal in mostmental diseases. An ESR is thus a useful help in findingout if a patient has diseases 01‘ a certain kind. The ESRalso helps us to know if a patient is getting better. Apatient’s red cells usually fall more slowly as he getsbetter.The ESR is probably one of the least useful tests inthis book. But it is sometimes wanted and is cheap andeasy to do. The important thing to remember is that theblood and the sodium citrare solurion must be mixedtogether in the right proportions. By this we mean thatthe right amount of blood must be mixed with the rightamount of sodium citrate solution.

The ESR 1 7.39make several of these bottlesand either keep them in therefrigerator or sterilize themin a pressure cooker7the ESR is21 mm in onehourbijoubottle18 2 ml pipette is beingfilled with blood/2 ml of blood are beingadded to the bottle which1 contains 0.5 ml of 3.8%sodium citrate 1a WestergrenWesteraren ESR tube isa\,being filled fiil”ed with blood1zlasma-top of cred cellaver020\000.5 ml. of 3.8%sodium citrate -I\the blood and the sodiumcitrateare being shaken4blood -4THETUBEhis blood hasinto sequestreneenslLNEAFTRACKFORXACTL’ANHOUR9DON’T MUDDLE THESE TWO THIS IS A WESTERGREN ESR TUBE0 20 40 60 80 100 320 940 160 180lfilength in mmTHIS IS A 2 ml PIPETTEFig. 7-33 The erythrocyte sedimentation rate or ESRMETHODTHE WESTERGREN ESR, FIG&E 7-331. Fill bijou bottles with 0.5 ml of 3.8% sodium citratesolution by the method described in Section 3.42a.2. lake blood into a sequestrene bottle.3. Measure 2 ml of the sequestrenated blood as describedin Seutions 1.17 and 4.6. Use a straight 2-mlpipette and put it into the sodium citrate bottle (4). Becareful not to muddle up a Wastergren tube and a 2-mlpipette. Look carefully at Pictures 9 and 10 and readwhat is said below.Instead of taking blood into sequestrene bottles, youcan, if you wish. take blood from the patient’s vein witha syringe and measure 2 ml of fresh blood straight intothe sodium citrate solution. But it is sometimes difficultto measure 2 ml of blood with a syringe, especially if airgets into the syringe with the blood. It is usually easierto put blood into a sequestrene bottle and measure 2 mlwith a pipette, as is shown here.5. Mix the blood with the sodium citrate solution.6. Fill a Westergren ESR tube in the same way as youfill a pipette. Fill the tube to just above the ‘0’ mark andlet the miniscus fall to the ‘0’ mark exactly.7. Put the tube in a rack. Take the time by a clock andwrite it down.8. Look at the tube exactly one hour later andmeasure how far the red cell layer has fallen. The tube islike a ruler and graduated in millimetres (mm). The centimetresare written as numbers. The number of mm thered cells have fallen in one hour is the ESR. The bestway to measure an hour is to use an alarm clock or aspecial timer.Don’t muddle a We$tergren tube with a 2-ml pipette.A Westergren tube is shown in Picture 9. It has a bluntend with a big hole in it and is marked from 0 to 180.These numbers are millimetres and measure length. Thered cell layer has fallen 21 mm in one hour in Picture 8,so the ESR is 2 1 mm an hour.

7 1 BloodA 2-ml pipette is shown in Picture 10. It has a pointedend with a small hole in it and is marked from 0 to 2.0.These numbers are millihvs or ml and measure volume.The top end of the tube is marked ‘ml’.7.40 The formol gel methodThis is another easy method. A drop of formalin is addedto a millilitre of the patient’s serum and the mixture isleft for 24 hours. If the serum is normal nothing happens.But, when the formal gel method is strongly positive(+ ++ +), the mixture of serum and formalin goes solidand white like the white part of a boiled egg. When theserum goes solid like this we say that it gels. The liquidserum has turned into a solid gel. Serum only gels whenit contains a greatly increased quantity of a kind ofprotein called globulm. Normal serum contains a littleglobulin, and in many diseases the globulin is slightlyincreased. Only in a few diseases, and only when theyhave lasted for several months at least, is there enoughglobulin for the serum to gel when formalin is added, andso give a positive form01 gel test. The form01 gel methodcannot by itself tell us which disease has caused thegreatly increased globulin in the serum. But in someparts of the world almost all the patients with a positiveform01 gel test will have one partidular disease. Forexample, in some areas of India a disease called visceralleishmaniasis (kala azar) is common. This disease oftencauses a positive form01 gel test. Therefore, if a patientin these areas of India has a positive form01 gel test,he probably has visceral leishmaniasis. The form01gel test is therefore a useful presumptive (probable)test for diseases of this kind in areas where they arecommon.METHODTHE FORMOL GEL TEST, FIGURE 7-341. Take some blood and put it into a clean, emptybottle.2. The blood will clot.3. Leave the clot until the next day. The clot willretract and clear, yellow serum will form.4. With a Pasteur pipette take about 1 ml of this clearserum and put it into a Kahn tube (6). The exact volumedoes not matter. You will soon learn what 1 ml lookslike in a Kahn tube.6. With a clean Pasteur pipette take some formalin(7) and add one drop to the 1 ml of serum in the Kahntube (81.9. If the test is very strongly positive (+++ +) theserum will gel (go solid) in as short a time as 20 minutes.If it is only just positive I+) it will not gel for 24hours. If the serum is still liquid the following day theformal gel test is negative.blood has been taken fromthe patient in this syringe -use formaldekNOT formolSalme7the blood is left to clot,and the clot is left toretract until next dayis beingi offserumclot - -blood is being put intoafter the clot has retracteda clean empty bottle there will be some clear serumlabout 1 ml of serum hasbeen but in this small tubegoes solid,goes whitesometimes(opaque)Fig. 7-34Theform01gel test

1”ig; I , _ :-!. * ip ’ _Make sure you use ‘Formaldehyde solution’ or‘Liquor Formaldehyde BP’: Don’t use formal saline. Theformalin in formol saline is too dilute for this test.7.41 The serum ureaThe methods for measuring the blood urea and the bloodsugar are both chemical methods for measuring substances in the blood. They are not likely to be needed inhealth centres; so the equipment for them has been put inthe main equipment list as being for hospitals only.In everybody’s blood there is a substance called urea.In healthy people there is always less than 40 mg of ureain every 100 ml of blood-less than 40 mg %. Urea ismade as the body uses up proteins and is excreted (gotrid of) by the kidneys. When the kidneys are diseasedurea may not be excreted properly and may stay in thebody. When there is too much urea in a patient’s bloodhe is said to be uraemie. A very uraemic patient mayhave a blood urea of several hundred mg %. Measuringthe blood urea is therefore a very useful way of findingout how a patient’s kidneys are working.Urea can be measured in whole blood, in serum or inplasma. The method described here uses serum orplasma. It is very easy. 0.1 ml (b ml) of serum or plasmaThe serum urea 1 7.41is put in the bottom of a graduated centrifuge tube. Twodrops of a urease solution are added. Urease is anenzyme which breaks down urea into ammonia andwater. Urease solution should be bought ready made, butit can be made from a special kind of bean (the jackbean), as described in Section 13.25. After about 5 minutes,when the urease enzyme has had time to work, areagent called Nessler’s solution is added. Nessler’s solutionmakes a brown colour with ammonia. The moreurea there was in the blood, the more ammonia will bemade and the deeper will be the brown colour whenNessler’s solution is added. This brownness is measuredwith the Lovibond comparator, the EEL calorimeter, orthe Grey wedge photometer. Nessler’s solution shouldalso be bought ready made, although it too can be madein a laboratory as described in Section 13.25.METHODMEASURING THE SERUM UREA, FIGURE 7-351. Take a clean graduated centrifuge tube. In the bottomof it put O-1 ml of clear serum or clear plasma froma sequestrene bottle. By ‘clear’ we mean that the serumshould be a clear yellow and not cloudy or red. Measurethis is a graduatedcentrifuge tubekeep the urease solutionin a small bottle with apipette in its cap, thebottles used for eye dropsare very good ones to use

1’.i 7 1 Bloodthe serum with the blood pipette ML 32. and fill thepipette to the mark which measures O-1 ml. If you havenot got a O-l-ml pipette you may use the O-l-ml graduationon the bottom of the centrifuge tube. but this willnot be accurate.Add two drops of ureas8 solution. Picture 1 showsthe serum and the unease solution in the bottom of thecentrifkge tube.Mix the serum and the urease solution by gently tappingthe bottom of the tube with your finger. Wait 5 minuteswhile the urease solution breaks down the urea andchanges it into ammonia.2. Using a Pasteur pipette fill the tube to exactly the8.9ml mark with water. This must be pure water-seewhat is said about this below. Again using a Pasteurpipette fill the tube to exactly the 9.5ml mark withNessler’s solution. The mixture will go brown. Hold yourthumb over the tube and turn it over once or twice sothat the solution is well mixed.Measure the depth of brownness of the solution withany of the following three instruments.THE LOVIBOND COMPARATORUse the Lovibond comparator exactly as in Section5.10. but use the blood urea discs 5/9A and 5/9B. This isprobably the best instrument to use with this method.THE GREY WEDGE PHOTOMETERUse the Grey wedge photometer exactly as describedin Section 5.14, but use the green No. 3 eyepiece.Change the number you read from the wheel of the Graywedge into mg % by using the scale in Figure 7-35.THEEEL COLORMETERUse the EEL calorimeter with the neutral greystandard exactly as described in Section 5.20, exceptthat you must use the blue filter llford 622. Find thereading on the galvanometer scale and then work outthe answer like this:Serum urea =Scale reading of test solutionScale reading with liquid grey standardX135mg%A graph can also be made for this method--seeFigure 5-9.There are several important things to remember. Thewater you use must not have any ammonia in it. Ordinarytap water often has enough ammonia in it to gobrown with Nessler’s solution, even without adding anyserum or ure8se. Distilled water should be used, butFootnote. As this book goes to press arrangements are in progress forrevising the scale in FIGURE 7-35. This will employ the same methodsdescribed here, but it is hoped that a new filter will make possible alonger scale for the blood urea. This will be incorporated in a pamphletissued with new Grey wedges, or available from the makers (KEE).some kinds of tap water may work. If tap water does notwork, try clean rainwater. To see if your water andurease solution are pure enough, do a ‘blank’ test likethis. Take a clean, empty centrifuge tube. Don’t add anyserum, but add two drops of urease solution and fill thetube to 8 ml with water. Then add Nessler’s solution to9.6 ml. The solution should stay almost as clear aswater. See what this blank test measures. If you find theblank measures more than about 10 mg % of ‘urea’, thewater is not pure enough, and you must find some waterwhich is pure and has less ammonia in it.Keep any ammonia or any ammonium salt (such asammonium sulphate) well away from this method or thereagents for it. This method will not work with plasmamade using ammonium oxalate as an anticoagulant.Keep the urease solution in a refrigerator all the time.If kept cold it will stay active for many months. If it isleft on the bench for too long the urease enzyme willsoon stop working.The Nessler’s solution must be made exactly as describedin Section 13.25. When Nessler’s solution isbought it must also have been made in this way. KeepNessler’s solution in a brown bottle. A brown depositwill form at the bottom of the bottle, but this does notmatter-use the clear supernatant solution on top of it.7.42 The blood sugarRead about the olood sugar and why we measure it inSection 8.6.Measuring the blood sugar is the most difficult of themethods described here. It has only been included becauseit is so useful in treating patients with diabetic coma.A measured amount of blood (0.1 ml) is diluted in ameasured volume of water. Two chemicals (sodium tungstateand sulphuric acid) are then added which willdeproteinize the blood (take away the proteins by precipitatingthem) and leave a clear supernatant fluidwithout any protein. A measured amount (2 ml) of thisclear protein-free fluid, which has in it some of the sugarfrom the blood, is then heated with an alkaline solutioncontaining copper. The sugar alters the alkaline copperso that, when a solution of phosphomolybdic acid isadded, the mixture goes blue. The more the sugar in theblood, the more will the alkaline copper be reduced andthe deeper will be the blue colour of the final solution.The depth of this blue colour is measured and tells us thepatient’s blood sugar.METHODMEASURING THE BLOOD SUGAR. FIGURES 7-36 AND 7-371. Put a tin of water on top of a tripod and bringit to the boil while yau are doing the first part of themethod.Take blood into a bottle containing a little potassium(or sodium) fluoride. This stops the blood cells using upthe sugar before the test-see Section 4.6.

,.,;+fi :_ . .2 The blood sugar 1 7.422 and 3. Fill a graduated centrifuge tube to exactlythe 3-5ml mark with water, preferably distilledtier.4 and 5. Using the blood pipette (ML 32) add 0.1 mlof blood to the water. Rinse the pipette once in theWater.6 and 7. Add O-2 ml of 10% sodium tungstat-eebelow for the best way to measure this.6. Mix by putting your thumb over the tube and turningit upside down.9. When you have finished mixing, dry your thumb byscraping it on the edge of the tube.10 and 11. Add 0.2 ml of 10% sulphuric acid.12 and 13. Mix and dry your thumb as before.14. The mixture will go brown.15. Let the tube stand for 10 minutes while the proteinforms a brown deposit with a clear supernatant.After this the mixture can either be centrifuged or filtered.If the mixture is to be centrifuged, centrifuge it as fastas you can for 5 mirrutes.16. If the mixture is to be filtered, filter it through asmallfilter paper. A Whatman No. 41 filter paper is best,but a Whatman No. 1 paper can be used.17. Using a 2-ml straight pipette take exactly 2 ml ofthe clear fluid you have got by centrifuging or filteringand put it in a clean test tube (18).19. Using another 2-ml straight pipette add 2 ml ofalkaline copper solution.20. Put a piece of cotton wool into the top of the tubethe blood pipette,to the 0.1 ml markblood pipettu1e 11u1e Ill mebottom of this pipette -------Iis exactly the samesize as the one for thesulphuric acid inpicture I I\0.1 ml of blood isbeing added to the /water to 3.5 mlthis pipetteshouldbe held uprightblood in a fluoridebottle: blood takenstraight from the earor finger can alsobe usedthis is the bottle of10% sodium tungstateL 4 drops u of sodium tungstatesolution being added tothe mixture of blood andwater in the tubeturn the tubeturn the tubeupside down10% sulphuric acid the mixture inside thetube turns brownFig. 7-36 The blood sugar- -onesteins

7 1 Bloodand put the tube into the tin of boiling water (21). Makesure the watar is boiling hard all the time and leave it inthe tin fiwfXACTLY 6 minutes. If the blood sugar is highthe mixture will go siightly brown.22. After exactly 6 minutes take the tube out ofthe boiling water and put it into a cup of cold water(23).24. Using another clean straight 2-ml pipette add 2ml of phosphomolybdic acid solution. The mixture willbubble and turn blue. Shake the tube gently to get rid ofthe bubbles.Measure the depth of the blue colour in the Lovibondcomparator. the Grey wedge photometer. or the EELcalorimeter.THE LOVIBONDCOMPARATOR26. Fill a Lovibond tube with the blue test solution.Use Lovibond discs 5/2A and 5/28 and follow the instructionsgiven in Section 5.10.THE GREY WEDGE PHOTOMETERUse the Grey wedge exactly as described in Section5.14 but use the red No. 3 eyepiece. Change the numberyou read on the wheel of the Grey wedge into the bloodsugar in mg % by using the scale in Figure 7-35.THE EEL COLORIMETERUse the EEL colorimetar with the neutral grey standardexactly as described in Section 5.20, except thatyou should use the red filter llford 608. Find the readingthe blue solution gives on the galvanometer scale andthen work out the answer like this:Blood sugar =Scale readinn of test solutionScale reading of neutral grey standard“145 mg %A graph can be made for this method like that inFigure 5-9.take aclearpipettefirst testtube&hY‘EIfI iclear filtrateIIiX ,IO9I07 65134clock022-take 2 ml ofmixture of filtrate \.filtrate and alkaline copperthe water from Picture 1will now be boiling10I, “L70B /76 513+add2mlof phosphomolybdicacid/’I water blanklet the tube boil forexactly six minutesLovibonddisc- bubble Of gas Sure there are nObubbles of gas on themlxrure goes bluesides of the tubeFig. 7-37 The blood sugar-twoI

,,:Measuring the plasma acetone with ‘Acetest’ tablets 1 7.43I There are many important details. One is the way in1 which the solutions of sodium tungstate and sulphuricacid are measured. One way is to use 2-ml pipettes.Another way to measure these solutions is to keep themin smal! bottles with teats and pipettes. See how manydrops from each I@ette are needed to fill an emptygraduated centrifuge tube to 1 ml. Divide the numberI of drops needed to make 1 ml by five. This will be thenumber of drops needed to measure O-2 ml and will beabout four drops. It is more imponuurt to add the sameamounts of sodium tungstate and sulphuric acid than it isto add exactly O-2 ml of each solution. Choose pipetteswith exactly the same-sized tips for both reagents.You may find it difficult to get enough clear supernatantor filtrate to make 2 ml. Centrifuge the mixturehard, and if you are filtering use a small paper. A largepaper will soak (drink) up too much ill&ate.The tubes must be exactly 6 minutes in water which isboiling very hard. You will get the wrong answer if thetime is wrong or if the water is not boiling hard.If you work in a small laboratory it is better to buy thesolutions or get them made in a big laboratory. Whereverthey come from, the solutions must be made exactly asdescribed in Section 13.25.‘Dextmstix’This is another method of measuring the blood sugarwhich is included as Choice 13 in Section 13.26. It is aspecial kind of very carefully made test paper. Althoughit looks easy to use, it must be used in exactly the rightway if it is to give the right answer. Even though ‘Dextrostix’and other paper tests look as if they are only piecesof paper, they are delicate (easily spoiled) instrumentswhich must be used exactly as the makers say.Store ‘Dextrostix’ in a cool dark cupboard. Don’tstore it in the sun beside a window or beside a sterilizer.Always keep the lid tightly screwed on, because dampspoils ‘Dextrostix’ quickly. Immediately you have takena ‘Dextrostix’ out of its bottle, screw on the cap. Try touse a bottle within 6 months, for it may not’ keep verylong. If you have several bottles, be careful to use theolder bottles first, and in this way to rotate (turn over)your stock.don’t move thepatient’s fingersideways likethisWRONG-- -.-.. ~_- .G---A=drop too small1-. ~--__RIGHTstart with a goodsized drop of blood-let the blood stay on the6paper for EXACTLY one minutehold the ‘Dextrostix’Juprightbottlelift the patient’s8 IMMEDIATELY comparewrth the colour chart ,/ .*/ don’t delay, or the/ colour will fadeMETHODTHE BLOOD SUGAR WITH ‘DEXTROSTIX1. Start with a good-sized drop of blood.2 end 3. Let the drop of blood touch the paper andstart timing one minute.4 and 5. Lift the patient’s finger off the ‘Dextrostix’ soas to leave behind a good-sized drop of blood.6. Let the drop of blood stay on the paper for exactlyone minute.7. Hold the ‘Dextrostix’ upright and wash off theblood with a short sharp jet of water from a wash bottle.Don’t hold it under the tap.8. Immediately compare the ‘Dextrostbc’ with the40 65 90 130 200colour chart, blood sugar in mg 0x1Fig. 7-38‘Dextrostix’colour chart. This will give you the blood sugar in milligramsper cent.7.43 Measuring the plasma acetone with ‘Acetest’tabletsIn Section 8.6 you will read how difficult it is to knowhow much insulin to give to a patient in diabetic coma.

7 1 BloodThe usual way to judge this is to measure his bloodsugar. But, when a patient’s blood sugar is greatly increased,the acetone in his blood is also increased. Thismeans that we can measure the acetone in a patient’sblood and use it to judge his probable blood sugar andhow much insulin to give him. The plasma acetone caneasily be measured by using ‘&etest’ tablets. You maybe asked to do the method, but the insulin will only begiven on the instruction of a doctor.METHODTHE PLASMA ACETONETake about 5 ml of blood from the patient and put it ina sequestrene bottle. Either allow the red cells to fall tothe bottom of the bottle by themselves, or spin the bloodto obtain some clear plasma.lake some of the clear plasma and put two dropsinto each of four clean test tubes. Label the tubes 1,2,3.4.Put two drops of saline into tube 2, six drops into tube3. and fourteen drops into tube 4. Tube 1 will containundiluted plasma; tuba 2 a 60% dilution of plasma; tube3 a 25% dilution; and tube 4 a 12% dilution of plasma.Test each dilution with an ‘Acetest’ tablet as exactlyshown in Picture A, Figure 82.Do the test every 2 hours with fresh plasma as long asthe patient is in coma.Wash the pipette wall in saline batween each dilutionof plasma.If there is a stronglv positive test (++++, dark blue)with the diluted plasma in tubes 3 or 4. the patient’sblood sugar is over 600 mg %, and he needs at least 100Units of soluble insulin. If the test is the same 2 hourslater, another 100 Units will be needed. When only thefirst tube gives a ++++ test, don’t give any more insulinfor the moment, but test again 2 hours fater.Test the urine at tho same time. Provided the bladder.is emptied each time, and the kidneys are working pro-., perly, the results of the urine test for sugar will reflect*. the blood sugar. and help to avoid the patient beinggiven too much insulin with resulting severe hypoglvcaemia.QUESTIONS1. How much haemoglobin has a healthy person?Why might you think that a patient had less haemoglobinthan this? List as many reasons as you can for a patienthaving less haemoglobin than normal.2. What is the most commonly used and also thecheapest treatment for anaemia? This treatment is onlysuitabi? for some patients. How could you find out whichpatients it is suitable for?3. Why will a patient who has suddenly lost muchb!ood not become anaemic immediately?4. What should a good thin blood film look like? Listsome of the things you should do in making a good thinblood film.5. When we count the white cells in a Neubauercounting chamber, we count the cells on two cornerblocks each of sixteen small squares and add two ‘OS’ tothe number of cells we find. Can you explain W/IJJ we dothis?6. Draw pictures of the different kinds of white cellsthat can be found in normal blood. List the variousthings that you would look for in distinguishing threedifferent kinds of white cells from one another.7. How are the red cells formed? What abnormal redcells might you find in a blood film?8. What kinds of abnormal blood picture do you knowand what diseases cause them?9. What is the difference between the sickle-cell traitand sickle-cell anaemia?10. What common kinds of anaemia do you know?How would you tell one kind of anaemia from otherkinds of anaemia?

8 1 Urine8.1 A clean specimen of urineUrine may seem the easiest specimen to get. Very often itis, and for many methods, sugar for example, any urinespecimen can be used. But for some methods, especiallythose for pus, protein, red cells, and bacteria, it is importantto get a clean specimen of urine. By clean we meanpure urine as it is in the bladder and without any dischargefrom the vagina or urethra added to it. Dischargesoften contain protein, pus, red cells, or bacteria. So, if wefind any of these in the urine, we cannot be sure if theywere in the urine iu the bladder, or were added to it in adischarge from the vagina, or the urethra. A specimen ofurine without any discharge is called a clean specimen ofurine. Clean specimens are taken in different ways inmen and women. In women clean urine is sometimestaken by putting a special rubber tube called a catheterinto the bladder. This is dangerous because bacteria maygo into the bladder on the catheter and infect, the urinarytract. The urinary tract is the kidneys which make urinefrom the blood, the ureters (the tubes joining the kidneysto the bladder), the bladder (the bag which holds theurine), and the urethra (the tube which joins the bladderto the outside world).METHODTAKfNG A CLEAN SPECIMEN OF URINEMENFetch a clean jar with a wide mouth or u specimenglass (Picture 3. Figure 6-l). If the urine is for culture,the jar must be sterile.Pull back the skin (the foreskin) eve; the patient’spenis (sex organ) and swab (clean) the opening (meatus)of the urethra with a sterile swab.Ask the patient to pass some of his urine. Throwthis away but catch some of the rest cf his urineinto the bottle or specimen glass. Any dischargein the urethra will have been washed awey by the firstpart of the urine, and the rest of the urine will be clean.This is sometimes called a mid-stream specimen orMsu.WOMEN (than must be dona by a nurse1Sit the woman down on a toilet with her legs wideapart. Fold back the lips (labial et the side of her urethra.Swab the opening of her urethra as in a men. Throwaway the first part of the urine and keep the middle part.PROTEIN, SUGAR, AND ACETONE8.2 Why we test the urineThe kidneys excrete (get rid of) water and many of thewaste substances in the blood that the body does notwant. This mixture of water and waste substances iscalled urine. One of the more important waste substancesis urea (see Section 1.10).Substances which are not waste substances sometimesgo into the urine. They go into the urine because somethingis wrong with the body or the urinary tract. One ofthe abnormal substances in the urine is the sugar calledglucose (see Section 8.6). When we are talking abouttesting the urine the words sugar and glucose usuallymean the same thing. Sugar in the urine is calledglycosuria. Glycosuria is found when a patient hasdiahetes.Protein is also found in the urine of some patientsproteinuria.It comes from the plasma into the urinewhen the urinary tract is diseased.We can easily test urine for protein and sugar. If muchsugar is found the patient probably has diabetes. If proteinis found, the patient has some disease oi’ his urinarytract.Don’t forget to look at a urine specimen as well astestmg it, and to report anything abnormal you may see.Urines are sometimes red with blood, or a deep brownishgreen with bile pigments, and they may also be colouredbv drugs.8.3 Testing the urine for sugar and proteinA few drops of urine are boiled with some Benedict’sreagent. Benedict’s reagent is a deep blue colour. If theurine is normal and there is no sugar in it, the Benedict’s

1_’8 1 Urinesolution will stay blue. But if there is sugar in the urine itwill change the colour of Benedict’s solution. The colourthat is formed depends upon how much sugar there is inthe urine. If the urine has a lot of sugar (++++) in it, themixture of urine and Benedict’s solution will changefrom deep blue to a deep red-brown colour like burntbricks. If there is not quite so much sugar in the urine(+++) the mixture will only go orange. If there is lesssugar still (++) the mixture will go yellow. If there isonly a very little sugar in the urine, the mixture will gogreen. As well as changing colour, the mixture of urineand Benedict’s solution also becomes turbid (cloudy),and, as it stands, a precipitate falls to the bottom of thetube. Some other substances besides sugar will changethe colour of Benedict’s solution, but we will not discussthem here.There are several things to be careful about when youdo this test. One is the volumes of urine and Benedict’ssolution you should use. Use much more Benedict’s solufionthan urine. Eight drops of urine and 5 ml ofBenedict’s solution are usually used, but it is bothquicker and cheaper to use smaller volumes of both urineand Benedict’s solution as described below.The sulphosalicylic acid test for protein is very easy.A few drops of 20% sulphosalicylic acid are added to theurine. If there is protein in the urine, the sulphosalicylicacid will precipitate it and make the urine cloudy. In theurine protein and sugar are often tested for together; sowe will describe both tests together.METHODTESTING THE URINE FOR PROTEIN AND SUGAR. FIGURE S-l1. Take two clean test tubes A and B.2. Fill tube A one third full with urine. In the Picturetuba A is shown being filled from a special urine specimenglass (31.4. Tip the urine in tube A quickly into tube B. Don’twait for tube A to drain completely, but put it back in therack. Four or five drops of urine will collect in thebottom of tube A (5).SUGAR6. Fill tube A with Benedict’s solution as deep as yourthumb is wide. it is best to keep the Benedict’s solutionin a wash bottle (7).8. Hold tube A in a piece of paper which has beenfolded into a strip as described in Section 3.11. Shakethe tube gently and boil it for a few minutes. Point thetube away from your face and hands in case the mixturejumps out of the tube. The mixture will be less likely tojump out if you shake the tube gently while it is boiled.Another way to stop the mixture jumping out is to boilonly the upper part of the tube, and to look for the colourchanges in this part of the tube only. The tube should beboiled for about 5 minutes, but most people usually boilit for about 2 minutes only. The best way of heating atube is to put it in a tin of boiling water for about 5minutes. Many tubes can be boiled at once in this way,but they must be labelled carefully, and you will need arack to hold them. This can easily be made of wire, orfrom a piece of wire netting. such as ‘chicken wire’.9. Put the tube in the rack and report the colour of theurine-‘red’ or ‘green’, or whatever its colour may be.Some people prefer to use the plus notation like this:PROTEINBlue -Green (no precipitate) 2Green (with precipitate) +Yellow . ++Orange +++Red ++++10. Add two or three drops of 20% sulphosalicylicacid to tube 8. If the urine is normal, nothing will happen(11 I. If protein is present the mixture will go milky(12). Report your answers with the plus notation (seeSection 4.4).Anothertest for proteinIf you have no sulphosalicylic acid you can test forprotein using dilute acetic acid and heat.METHODANOTHER TEST FOR PROTEIN IN THE URINEFill a test tube nearly to the top with urine. Hold thetube by the bottom and heat only the top part of theurine in a flame until it boils. Take the tube out of theflame and add two or three drops of 3% acetic acid. Ifthe urine becomes turbid when it is heated and staysturbid when acid is added, the test for protein is positive.The test is also positive if the urine stays clear whenheated but goes turbid when acid is added. But the testis negative if acid removes the turbidity made by heating.This turbidity made by heating and dissolved by acidis caused by phosphates and not by protein and isnormal.Measuring protein in the urineA common way of measuring the amount of protein inthe urine is to use a method called Esbach’s method. Thishas not been described here because you can measurethe urine protein in the same way that you can measurethe CSF protein. Use the proteinometer standard, or theGrey wedge photometer as described in Section 9.13.Protein in the urine is usually measured in grams perlitre and not in mg % like the CSF protein. But it is easyto change one way of measuring into the other. 100 mg %(100 mg in 100 ml) is the same as 1 g (10 times 100mg, 1,000 mg, or 1 g) in one litre (10 times 100 ml,1,000 ml, or 1 litre). If there is so much protein in theurine that it goes beyond the end of the scale ( 100 mg %

“:. * -,y,: .&$.,i.:, .’ ; I 1 I . ! I~“-~gjl ‘.with the proteinometer standards, or about 150 mg %with the Grey wedge), you may have to dilute the urinebefore you add the sulphosalicylic acid. @, for example,you dilute 1 ml of urine with 1 ml of water, you will havediluted the urine in twice its volume (1 ml into a total of _2 ml). You will ‘therefore have to multiply the answer you .get by two. If there is much protein in the urine you mayhave to dilute it even more.8.4 The meaning of proteinuriaMany diseases cause proteinuria. Here are some of themore important ones.FeverThe meaning of proteinuria 1 8.4Many patients with a high fever have a little protein intheir qrine. When their fever stops their proteinuria stopsalso. This is a com?on cause of mild proteinuria.Bacterial infection of the urinary tractUrinary infections are common in women. When bacteriagrow in the urinary tract and infect it, there isusually protein in the urine. A patient with an infectionof the lower part of his urinary tract usually passes hisurine more frequently than normal (frequency). He hasthis is urine in a specimen gl3rclean testtubes, Aand84 4$guy,4urine&;I, 4 ‘4.I.-.II’i,;” I\!1fill tubeA as deep asyour thumbwithBenedict’ssolution3of urine willSugar10a :clear11’1 II ’bluegreen(no precipitate) 2green+(with precipitate)brownorangeredE+++++POSITIVEturbidor cloudyFig. 8-l Testing the urine for sugar and protein

often to pass it in a hurry - (urgency) - and usually has painon pass& it (dysacia). When a patient has an infectionof the upper part of his urinary tract (his kidneys and theupper parts of his ureters) he may have no urgencv.fie&&y, and dysuria, but only fe&r, and perhaps pa&in his kidneys. When a patient has any kind of urinaryinfection, protein and pus cells can usually be found inhis urine (pyuria, pus in the urine). These pus cells arepolymorphs (see Section 7.14) that have come out of theblood to fight bacteria invading the urinary tract. Theycan’ easily be found and counted by looking at the urinewith a microscope (see Section 8.11) and are very usefulin diagnosing urinary infection. Bacteria may also beseen when infected urine is looked at with a microscope.Urinary infections can often be treated quite easilyat a health centre; so it is important to be able todiagnose them. Patients are usually given tablets of asulphonamide drug to kill the bacteria in their urinarytracts.Schiktosomiasis(bilhania or bilharziasis)As you will read in Section 8.15, there is a worm calledSchistosoma haematobium which lives in the veins in thewalls of the bladder and ureters. The urine of infectedpatients usually has protein and red cells in it besides theova of S. haematobium. In areas where schistosomiasis iscommon, it will be much the commonest cause of proteinuria.If schistosomiasis is common where you work,always look for the ova of S. haematobium wheneveryou find protein in the urine.Vaginal and urethral dischargesWhen patients have infections of the vagina (a woman’sbirth passage) and urethra they have discharges fromthese organs. A discharge is an abnormal liquid and isoften pus. These discharges contain protein and maycontain pus cells and red cells. If vaginal and urethraldischarges get into the urine, patients may seem to haveproteinuria, pyuria, or baematuria (blood in the urine)when really they have not. If you find protein, pus cellsor red cells in an ordinary specimen, it is often necessaryto take another specimen of urine and this time to makesure that no urethral or vaginal discharge gets into it.Taking a clean specimen of this kind is described inSection 8.1. Blood often gets into the urine during menstruation.Toxaemiaof pregnancyThis is a disease which some pregnant mothers get. Theirlegs swell, they get protein in the urine, and their bloodpressure (the pressure of blood in their arteries) getsabnormally high. If the toxaemia gets very bad them~!kor nap hsve fits .md her baby may die. One of thet:d-ii~si stgm ?i' f;hu~ ,.,i., oJ*‘yregnancy is 1.; L;;I .:;,; -:;-urine. This is why ati mothers must have tireir urinetested for protein at every antenatat visit.NephritisSome kinds of kidney disease (nephritis) are L less common,but important cause of proteinuria. In nephritisthere is often much more protein in the urine than thereis in either fever or in urinary infections. There are manykinds of nephritis, but we will describe only three, acutenephritis, chronic nephritis, and the nephrotic syndrome.AcutenephritisThis is a short-lasting condition which often followsabout 10 days after a sore throat. There are red cells andgranular casts (see Section 8.13) as well as protein in theurine. There may be so many red cells that the urinelooks quite red. The patient’s blood pressure and bloodurea are often raised, and his face and ankles may beswollen with fluid in the tissues (oedema). Most patientsget well in a few weeks. A few patients go on to havechronic nephritis.ChronicnephritisThe word chronic means long-lasting, and these patientsusually have their kidney disease for a long time. Theymay not know they are ill, and many cases of chronicnephritis are only found on routine urine testing. Evenso, after feeling fairly well for some months or years, theblood pressure and blood urea of these patients rise, andthey die.Patients with chronic nephritis have protein andgranular casts in their urine.NephroticsyndromeThese patients have much protein (+ + + or + + + +) intheir urine and too littie in their blood, because theirkidneys let much protein leak from the blood into theurine. Their legs become swollen with fluid-they aresaid to become oedematous. Some casts may be seen inthe urine, but the most obvious signs are the severeproteinuria and the oedema. These patients may be quitecommon, most of them are children.HeartfailureWhen the heart cannot pump the blood round the bodyproperly, the patient is said to have heart failure orcardiac failure. Most patients with heart failure have alittle protein in their urine. But patients with heart failureare not usually common: so you will not find this to be acommon cause of proteinuria.Diseases which do not have proteinuriaC!hi:+e~~ with kwashiorkor and severe hookworm-..*&I ...--‘. ....- L. AC r : -- hays pe-+n,ri-~. 7 ills I; ;IKJ&!, &p:scthere ri~aq by GC&na m both these diseases. A patientwith oedema and heavy proteinuria is therefore unlikely

;,‘-j_‘. ..‘I I: ^I .p-: 1.’‘,_i_.,,I :- ,.Iy,. _ ; “r,;- ;‘ L;““,,x / ,, q,: ,,‘I , .‘a I( \ ;:, -:;,F ./ ..,.’ -,_, +.;-.., “( .- ,.+; (.-.Routine urine testing 1 8.5to have kwashiorkor, or severe hookwol m anaemia. Heis more likely to have nephritis or the nephrotic syndrome.8.5 Routine urine testingWhen a patient is examined and his history is taken,there may be no reason for thinking that he has diabetesor that he has any disease of his urinary tract. Therefore,unless a patient’s urine is always tested for sugar andprotein, diabetes and disease of the urinary tract mayeasily be overlooked. By overlooked we mean that thesediseases will not be diagnosed when they should be.Therqfiore, the urine of all patients who are ill enough tobe in hospital must always be tested for sugar and protein.Some kinds of outpatient should also have theirurine tested routinely. By routinely we mean always oras a general rule. It is easy to test the urine routinelybecause the tests are quick, simple, and cheap. Test theurine of the following patients routinely for protein andsugar:1. All patients in the wards, especially those who arevery ill.2. Any outpatient with the following symptoms:loss of weight, great thirst or passing too much urine(suggests diabetes), frequency, urgency, dysuria (thesethree suggest a urinary infection), oedema (suggestsnephritis or heart failure).3. Patients with the last four symptoms are likely tohave only proteinuria, but it is best to test routinelyfor both protein and sugar.Whenever you Jind protein, examine the spun depositfor red cells, pus cells, casts, and the ova of S. haematobiumas described below. If necessary. count the puscells.Whenever you find much sugar (+ + or more) test theurine for acetone. These methods are described in thenext sections, but, before you can understand why theyare used, you must learn more about diabetes.8.6 Diabetes and the blood sugarWe all have some sugar in our blood. This sugar is calledglucose, and it is different from the sugar we put into ourtea, which is called sucrose. There is more sugar in ourblood soon after a meal. This is because part of our food(the carbohydrate part) is made into sugar in our gut(stomach, intestines) and absorbed (taken into ourblood). Some hours after a meal there is less sugar in ourblood, because some of it has been used or stored by ourbody. The blood sugar measured at this time is called thefasting (without eating) blood sugar. The fasting bloodsugar of normal people is between 65 and 105 mg per100 ml. But, about an hour after we have had a meal ourblood sugar may rise as high as 150 mg per 100 nil.,Because meals alter the blood sugar so much, WC UN&try to measure the fasting blood sugar. The easiest timeto measure it is in the early morning, when a patient hashad no food since the evening before and no breakfast.In the disease called diabetes the cells of the body areunable to use sugar properly, and there is too muchsugar in the blood (hypcrglycaemia: hyper = too much,glyc = sugar, aemia = blood). A diabetic may have 500mg % and more of sugar in his blood. Because there is somuch sugar in the blood, some of it is excreted (goes outof the body) into the urine. This is why we test the urinefor sugar. If we find sugar in a patient’s urine, he probablyhas diabetes. We can also see if a patient hasdiabetes by measuripg his fasting blood sugar. This issometimes useful, but it is not as easy as testing his urine.A patient may have diabetes if his fasting blood sugaris more than 105 mg 96. If a diabetic patient is nottreated he may get thin and weak. He will be thirsty. hewill drink much water, and he will pass much urine.His body may become dehydrated (de = without,hyd = water, lacking water), and he may go into a comaand die. A patient in a coma seems to be asleep butcannot be woken up. This kind of coma is called diabeticcoma or hypcrglycaemic coma.If the high blood sugar of a diabetic can be lowered tonormal he will stay well. This is usually done by givinghim injections of a drug called insulin once or twice aday for the rest of his life. Some fat patients with milddiabetes get better when they eat less, and other diabeticsare given tablets. If a diabetic is being treated withinsulin he needs just the right amount of insulin to bringhis blood sugar to normal but not below it. If a diabetic isgiven too much insulin his blood sugar will fall too low.and he will have hypoglycaemia (hypo = too little).A hypoglycaemic patient feels ill and weak and is coldand sweaty. If a diabetic is given too much insulin hisblood sugar may go so low that he may go into anothersort of coma and may die, unless he is quickly givensome sugar. This kind of coma is called hypoglycaemiccoma.Diabetics differ from one another, and each diabeticneeds different treatment. That is, they need differentamounts of insulin, starving or tablets if their bloodsugar is to be brought down to normal but not below it.It is too difficult to measure the blood sugar of a diabeticeach time we want to see if he is being properly treated.Instead, we test his urine for sugar. If the urine of adiabetic is blue with Benedict’s method, his blood sugaris probably too low. If it is green, his blood sugar isabout right. But, if his urine is yellow with Benedict’smethod, and especially if it is red, his blood sugar is toohigh. It is seldom possible to treat a diabetic so carefullythat his urine is always just green, but this should be theaim.As you have read, the blood sugar is low before mealsand high soon afterwards. The dose of insulin is adjustedso that a specimen of urine passed just before meals is‘green’ or ‘blue’. It does not matter if the urine is ‘yellow’or ‘red’ afler meals. Benedict’s method is thus a usefulit;;y J. +- LiLn-. ?’ il”.!+p din& -‘PC j .~L scd f?f sdjmtir?o. the wav it istreated.

i8 1 Urine8.7 Acetone and sodium nitroprusside and is described in Section3.39. Using a spatula (2). put a small quantity of powderin a test tube. say just enough to cover the round bottomof the tube (3).As you have read, if patients with sever.% diabetes are nottreated, they may first become sleepy and then go into acoma. These patients have a very high blood sugar andpass much sugar in their urine. They also pass a substancecalled acetone in their urine, and breathe it out intheir breath. Diabetic coma is difficult to treat, andpatients sometimes die. It must therefore be prevented.Acetone in the urine is a sign that the patient is about togo into a coma and needs treatment quickly. The urine ofa diabetic must be tested often. If it is yellow or red byBenedict’s method, AL WAYS TEST FOR ACETONE.If you find acetone, the patient needs treatment’ quickly.If the patient is already being treated with insulin, butstill has acetone in his urine, he is not getting enoughinsulin, and the dose must be increased.Patients in a diabetic coma need much insulin. It issometimes difficult to know how much insulin to givethem, and it is useful to know what their blood sugar isduring treatment. This is the main reason why a methodfor measuring blood sugar has been described in Section7.42.Acetone is sometimes found in the urine withoutsugar, particularly after a patient has been vomiting andespecially in children. It may also be found if a patienthas been without food for a long time, or in women whoare very tired after spending a long time in labour (givingbirth). There is seldom much acetone in the urine of thesepatients and it has nothing to do with diabetes.There are several ways of testing the urine for acetone.The simplest way uses ‘Acetest’ tablets which are in themain equipment list. Two other methods will also bedescribed. One is the standard (usual) Rothera methodusing ammonia. The other is an altered Rothera methodwhich uses sodium carbonate instead of ammonia. It ismuch the best.METHODTESTING URINE FOR ACETONE. FIGURE &2A. WITH ‘ACETEST’ TABLETS4. With a Pasteur pipette of urine (4). just moisten thepowder at the bottom of the tube. Three or four drops ofurine will probably be enough (5).6. Wait one minute-time it if possible with yourwatch. Hold the tube against a sheet of white paper, sothat any colour change can be seen more easily.7. If there is no acetone in the urine, there will be nochange in colour.8. If there is acetone in the urine, a purple colour willbe seen. If there is much acetone in the urine, a deeppurple colour will form rapidly.There are various ways of reporting the colour. Here isone which can be used both with this way of doingRothera’s test and with the next one:No colour change -Very slight purple colour tDefinite purple colour +Slow-forming medium purple + +Slow-forming deep purple +++Rapid-forming deep purple ++++C. STANDARD ROTHERA METHOD1. The standard Rothera’s mixture contains sodiumnitroprusside and ammonium sulphate-see Section3.39.2. Use a spoon or spatula to fill the bottom half inchof a test tube with Rothera’s mixture (3).4. Just cover the layer of powder with urine (51.6. Shake gently. Most of the powder will dissolve inthe urine. which will become saturated with ammoniumsulphate.7. Add strong ammonia. This must be 22% or 25%ammonia with a specific gravity of 0.92 or O-88. Theexact amount of ammonia to add is not important, buttry to add about a third as much ammonia as urine (8).9 and 10. Mix, and report the colour change exactly asin Method 8 above.1. Put an ‘Acetest’ tablet on a piece of clean whitepaper.2. Put one drop of urine on the tablet.3. Wait for half a minute (30 seconds) and look at thecolour. If possible use a watch to measure 30 seconds. Ifthere is no acetone in the urine, the tablet will stay whiteor be coloured yellow from the urine (4). If there isacetone in the urine, the tablet will change colour. Ifthere is only a little acetone (+) it will go a pale purplecolour. If there is much acetone in the urine (+ + + + ) thetablet will quickly go a deep purple colour (5).6. MODIFIED ROTHERA METHOD1. The modified (altered) Rothera’s reagent used hereIs d r:t:*ure of amt?leni**-- . . . . l , ar!*: ---‘-G-*- .tLl..T; sodium carbonate,BILE PIGMENTS8.8 Jaundice and some tests for bile pigmentsA pigment is a coloured substance. The liver does manyjobs for the body. One of them is to make the fluid calledbile and send it into the gut along tubes called the bileducts (tubes). Bile is a thick greenish-yellow fluid whichcontains many substances. One of them is a yellow substancecalled bilirubin which helps to give bile its yellowcolour. Bilirubin is formed from the haemoglobin ofbroken down (lysed) red cells. In some diseases the yellowbilirubin does not get into the gut as it should, butstays in the blood which then has an abnormally highconcentration of bilirubin. When this happens, somebilirubin gets out of the blood into the rest of the body

,- ;>. .-:I_. -.._). Jaundice and some tests for bile pigments1 a.8ABC:Jehite/Acetest tabletpaper:lean testtube73gi$LcfFu3 WAIT 30 SECONDS_-._ ..-.x3;‘:,4just wet the powderat the bottom ofthe tube with urineb 6the powder1 :ammoniauNEGATIVEMtNUTEPOSITIVEtlw rill dissolveif I th’e urinePOSITIVEno c&ourpurpleand stains it yellow. When a person is stained yellowwith bilirubin in this way he is said to be jaundiced(icteric). It is easy to see when white-skinned people arejaundiced, but jaundice is not so easy to see in darkskinnedpeople. The best way to see if a dark-skinnedperson is jaundiced is to look at the white part (sclera) ofhis eyes which stain yellow with bilirubin.In diabetes there is so much sugar in the blood thatsome leaks out into the urine. In jaundice, too, there is somuch bilirubin in the blood that some leaks into the urineand can be tested for with Fouchet’s test (spoken‘foushay’). You may say. why test the urine for bilirubinto see if a patient is jaundiced, if it is so easy to look athis eyes (or his skin if he is white)? The answer is that wecan often find bilirubin in the urine before a patient’seyes become obviously yellow. This is especially usefulin the early days of a liver disease called infectious hepatitis(hepatitis means inflammation of the liver). Sometimesalso there is doubt as tn whether a patient’s eyesFig. 8-2 Testing the urine for acetoneare yellow or not. This doubt can be settled by testing hisurine for bilirubin.There is another bile pigment in the urine which it iseasy to test for. This is urobilinogen, which we test forwith Ehrlich’s test. Bacteria in the large gut make urobilinogenfrom bilirubin. Urobilinogen is partly absorbedfrom the gut into the blood and excreted in the bile. Aswith sugar and bilirubin, if there is more urobilinogenthan there should be in the blood, some of it will go intothe urine.There are two main reasons for increased urobilinogenin the urine.1. When red cells are being haemolysed extra fast (insickle-cell anaemia or malaria for example), muchhaemoglobin is produced. Much bilirubin is formed fromit and passes into the bile. Much urobilinogen is formedfrom this bilirubin in the gut. Some of this is absorbedinto the blood stream and excreted by the kidneys intothe i!f!y. !P +I. ...Prefore much urobilinogen is found in the

;gg;;r-,,“‘, ;i II;.‘8.( “tine, . _ .F ~-NEGATIVE not shown, the precipitateis white or pale yellow and thereis no blue colour with Fouchet’s1Mzc?2filter paper taken out of thefunnel and put in a sink oron a white tile or plater- .-,.. -,..,ttube l/3 full tuba baing filled clear filtrateof urine nearly to the top not wantedwith 10% bariumchloride solutionurine it may mean that there is increased destruction ofred cells. If a patient is anaemic and has much urobilinogenin his urine he may have a haemolytic anaemia (seeSection 7.9).2. Urobilinogen is secreted into the bile by the cells ofthe liver. When the iiver cells are diseased they may notbe able to excrete urobilinogen as they should. Urobilinogentherefore increases in the blood and goes into theurine. An abnormal amount of urobilinogen in the urinemay mean that the iiver is diseased.Nothing more will be said about the meaning of thesetests here. They will mostly be asked for by doctors inhospitals who will understand their meaning.Look at the colour of the urine. The pale yellowcolour of normal urine is not due to bile pigments but toother substances. It is not possible to tell the yellowcolour of a concentrated (strong) normal urine, from thecolour due to small amounts of bile pigments. This is whywe have to test it. But, if there is much bile in the urine, itis easily seen to be abnormal, for it will be a dark brownishgreen with yellow bubbles (foam) when it is shaken.METHDDFOUCHET’S TEST FOR BILiRUBIN. FIGURE S-31. Take a test tube one-third full of urine.2. Fill it nearly to the top with 10% barium chloride. Itwill go milky. with a white or yellowish precipitate.3. Pour the milky mixture through a small filter paperLFig. 8-3 Fouchet’s test for bile pigmentsdeep yellow POSlTiVE‘precipitateblue colour with,. I Fouchet’s reagentheid in a small funnel. If you don’t know how to fold afilter paper, look in Section 3.11. When all or nearly allthe liquid (filtrate) has passed through, take the paperout and lay it in a sink or on a white tile. If the patient isjaundiced, the precipitate will be yellow; otherwise itwill be white or nearly whiie.4. Add two drops of Fouchht’s reagent (see Section3.30) to the precipitate on the paper.5. If bilirubin is present the precipitate will go bluegreen,either a pale blue-green (+ 1 if there is only a littlebilirubin present, or a darker blue-green (+ + + + 1, if thereis much bilirubih there.6. If the test is negative there will be no blue colour.METHODEHRLICH’S TEST FOR UROBILOGEN. FIGURE S-41. Fill a test tube to the depth of two fingers withfresh urine. There will be 5 ml in the tube, and it will beabout one-quarter full. The urine must be fresh. If theurine is more than about an hour old the test will notwork.2. Add an aqua1 amount of Ehrlich’s reagent. The tubewill now be about half full.3. Wait 10 minutes.4. Fill the tube nearly to the top with a saturated solutionof sodium acetate.5. If there is only a normal amount of urobilinogen inthe urine it will be yellowish in colour.

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Testingthe urine for sulphones1 8.10dip the strip into the, ,LPOSITIVE, the strip will go ‘NEGATIVE, the strip willblue if the patient has been stay the same colour if thetaking PAS or any other patient has not been takingsalicylate drug PAS or any other salicylatedrug,these are the PAS test stripsmade by the method describedin Section 3.38Fig. 8-6 Testing the urine for PASIf a red colour forms within one minute, the patient islikely to have taken INH up to 12 hours before.If the urine only goes pink within one minute, it islikely that the patient took his INH between 12 and 24hours previously.If the urine remains yellow, the patient has not takenany INH during the previous 24 hours.the urine. Some other drugs such as aspirin and theother salicylates also give this colour.5. If the strip does not go brownish purple, there is noPAS (or aspirin or other salicylate) in the urine.8.10 Testing the urine for sulphonesSulphones, such as DDS (Diamino Diphenyl Sulphone),There may be many urines from the tuberculosis are drugs used to treat leprosy. It is often useful to knowclinic to be tested. When there are, number the specimensand number the holes, as shown on the tile inif leprosy patients are taking their sulphones, and thefollowing method is a very easy one.FIGURE 8-5. First put the four drops of each of the urinesin the tile into their right holes. Next add the four dropsof cyanide, then the nine drops of chloramine T.METHODThis test will only work with INH that has beenURINE SULPHONES. FIGURE 8-7through the body and has been altered by it. It will notwork with a solution of INH tablets.1. Take3.43a.one of the papers prepared in Section2. Put a drop of urine in the middle of the paper.3. If the test is positive, there will be a strong yellowMETHODring within 10 seconds. If the yellow ring comes after 10TESTING THE URINE FOR PAS. FIGURE 6-6seconds, it is not due to sulphones.1. Make PAS test strips as described in Section4. If the test is negative, there is no yellow ring.3.38.2. Take hold of a paper by one end. Don’t make the papers too small, or the drop of urine3. Dip the other end of the paper into the urine. too big. If you do, the coloured ring will go over the edge4. If the paper goes a brownish purple, there is PAS in of the paper. By this method sulphone can be found in

8 1 utin? _ _/the urine 60-90 minutes after 100 mg of DDS has beentaken by mouth. This method is usually only positive ifpatients have been taking their sulphones regularly indoses of not less than 50- 100 mg daily. If, however, theyhave been on these doses for a very long time, sulphonesmay be found in their urine for up to a month after theyhave stopped treatment.MICROSCOPYOF THE URINE8.11 Pus cells in the urineInfection of the urinary tract was discussed in Section8.4. You learnt that, when the urinary tract is infected,right way to do this. A Fuchs-Rosenthal chamber isbest, but a Neubauer chamber can be used.4. Fill a Pasteur pipette with the urine. Fill the countingchamber.5. If a Neubauer chamber is being used, count thecells in one corner block of sixteen small squares andmultiply by ten.8. If a Fuchs-Rosenthal chamber is being used, countthe number of pus cells in one corner block of sixteensmall squares and multiply by five. In this way you willget the number of pus cells in 1 cu mm.7. Some of the more important things to be seen inthe urine are drawn in this Picture, which shows ahigh power view of urine in a counting chamber. Pus10 SECONDS LATERpatient’s urine --NEGATIVE, no yellow ring, oronly a faint one at the edge ofthe spot of urinespecial filter paper asmade in Section 3.43aif a yellow colour comes after 10 secondsIt is not due to sulphonesFig. 8-7 Testing the urine for sulohonesprotein, bacteria, and pus cells are usually found in theurine. Finding pus cells in the urine (pyuria) is a muchmore certain sign of urinary tract infection than findingprotein. It is important therefore to be able to look at theurine for pus cells. This is easily done. Put a drop of theurine in a counting chamber, and then count the cells init just as if you were counting cells in the blood (seeSection 7.29) or the CSF (see Section 9.9).METHODPUS CELLS IN THE URINE, FIGURE S-81. Get a clean or mid-stream specimen of urine in auniversal container as described in Section 8.1.2. Mix the specimen well by shaking the bottle withthe lid on. If the urine is not well mixed, all the pus callsmay stay at the bottom of the bottle.3. Put a coverslip on a counting chamber-look atSection 7.29 and Picture 2, Figure 7-21, to learn thecalls are larger than red calls. They have many granulesof different sizes which may look faintly greenish.Several segments of the nucleus can usually beseen, and there is often a clear area at the edge of thecell. Red cells are faintly yellowish-red; they are oftencrenatad and have short points sticking out of them(sea Section 7.25). Epitholial cells are much largerand flatter. They usually have a nucleus that is easilyseen.If at first you have difficulty in being sure about whatblood cells and pus cells look like, put a loopful of bloodor pus in a drop of urine, and look at it with a microscope.8.12 What it means to find pus cells in the urineIn some countries healthy men seldom have any pus cellsin their urine. In other countries a mild infection of theurethra (urethritis) is so usual that to have some pus cells

_/Looking at the centrifuged deposit 1 8.13Of urinel A universalp-y-,containerIir.-1--h‘II 0I --t-imix wellHIGH POWER MICROSCOPEVIEW5 NEUBAUER RULING.I6 smallx lO=cells percu mmof urinesquares6 FUCHS ROSENTHAL RULING.16 smallx5=‘cells persu mm‘of urine1 segments of ‘ihe’- w.!Fig. 8-8 Counting pus cells in the urineepithelial’ccl Iruling on thecountmgchamberin the urine may be more common than to have none. Afew pus cells are also often found in the urine of healthywomen everywhere. Because of this, it is usual to takesome figure for the number of pus cells in the urinebelow which patients are said to be normal, and abovewhich they are said to be abnormal, and to have aninfection of their urinary tracts. Fifty pus cells per cumm is a common figure to take, Patients with less than50 pus cells per cu mm are thought to be normal.Patients with more than 50 pus cells per cu mm arethought to be abnormal, and to have a urinary tractinfection. This is only a guide, but it is a useful one. Insome countries IO pus cells per cu mm is taken as theupper limit of normal.8.13 Looking at the centrifuged depositIn the last method, when we counted pus cells in theurine, we took care to make sure that the urine was wellmixed. We looked at the urine itself and not the deposit.In this method we look for several things in the spundeposit of urine.METHODEXAMINING THE CENTRIFUGED DEPOSIT OF URINEMix the specimen of urine.Fill a centrifuge tube with urine.Centrifuge for about 3 minutes with an electric centrifuge,or about 5 minutes with a hand centrifuge.Pour away the supernatant urine.Using a Pasteur pipette suspend the deposit in thelast drop of urine and put it on a slide under e coverslip.Search the whole area of the coverslip with a low powerobjective. Then look at some fields with a high powerobjective.Be careful not to pour away the deposit as you pouraway the supernatant ,urine. If a hand centrifuge hasbeen used and the deposit is not well packed at the

ottom of the tube, pour away only the top part of thesupernatant Remove the last inch of supematant urinewith a Pasteur pipette, and mix the sediment with thelest drop or two of urine.Some of the things that can be seen in a urine deposithave been drawn in FIGURE 8-10. They have all beencarefully drawn ‘to scale’. That is, the; have all beendrawn the right size compared to one another. To showhow big things are a line has been drawnat the bottom ofthefigure and marked in Flrn (see Section 6. lb). Beside it is ared cell, which you will remember is about 7-S btrn across.One of the most important things to look for are casts.CastsIn the kidney there are millions of tubules (small tubes)through which the urine goes on its way to the bladder.These tubules are shown in Picture A, FIGURE 8-9. Inmany kinds of kidney disease these tubules becomeblocked with protein or the remains of dead cells (PictureB). Very often the solid substance blocking the tubulebecomes loose and goes down the tubule and into theurine (Picture C). This material which was blocking thetubule and has come loose is called a cast. Because it wasformed inside a tubule a cast has the shape of the tubulefrom which it came. Casts are usually shaped like the onedrawn in Picture D. This is a diagram of a cast, and somedrawings of casts can be seen in FIGURE 8- 10.Casts are seen in several kidney diseases, and there areseveral kinds of cast. To make things simple we shallconsider only two kinds of cast-hyaline casts and granularcasts. Hyaline (like glass) casts are clear or transparent(light can shine through them). They either have nogranules in them or only a few granules. Normal urineoften has some of these hyaline casts, and they do notmean that the kidney is diseased.As their name says, granular casts are made of coarsegranules. A granule is something very small and roundlike a small seed. Granular casts are not found in normalurine, and if they are found in the urine they mean thatthe patient has diseased kidneys. Very often this diseaseis serious. Granular casts are only found when there isprotein in the urine. All urines in which protein is found(+ + or more) should be centrifuged, so that the depositcan be searched for casts under a microscope. The findingof protein and granular casts may mean that a patienthas chronic nephritis (see Section 8.4).Two sorts of casts are shown in FIGURE 8-10: somegranular casts in Picture 10 and some hyaline casts inPicture Il. The granules in Picture 1Oa are especiallybig. Casts often look as if they are partly empty inside asin Picture lob. In Picture 1Oc there are the remains of anucleus. The end of the cast in Picture 10d is twisted(turned) on itself and has nearly broken off.The cast in Picture 1 la is very nearly completelyhyaline-that is, it is clear like glass. Those in Pictures1 lb and 1 Ic have many tiny granules, but even so theseare very different from the big dark granules of the truegranular casts in Picture 10. Nevertheless casts aresometimes seen which are partly granular and partlyhyaline. If in doubt it is best to report them as granularcasts. Other kinds of casts are also seen-cellular, waxy,and pigment casts; but these are not so common, andthey will not be described here. It is probably best if youreport all casts which are not hyaline as granular casts.Other findings in the centrifuged deposit-Figure8-70The first four pictures in FIGURE P- 10 show the ova ofSchistosoma haematobiurrl which are described in moredetail in Section 8.15.The next pictures show various kinds of cell. Red cellsurineis madethese are normalkidney tubulesthis tubuletowards theurinary bladderthis tubule isblocked bysubstances inthe urine orcoming frominjured cellsFig. 8-9Castsis!;’ _.

aliSCH!STOSOME OVA 3deadSchistosoma haematobium / iwhen thePlate 76THlS IS A DRAWINeverything is drawnapproximately to scale5 REDCELLS 6freshold7 PUS CELLS 8 9 EPITHELIAL CELLScrenateothe cell membranes areleft and the haernoglobinhas gonePlates 57 & 64.

8 1 Urineare drawn in Pictures 5 and 6. The red cells in Picture 5are fresh and would look slightly yellowish-red. Thethree cells at ‘a’ at the bottom of Picture 5 are slightlycrenated (see Section 1.18), and their edges, instead ofbeing smooth. are slightly rough. Crenated red cells arealso drawn in FIGURE 7- 15. In this figure they are drawnlarger and their shape will be understood more easily.The red cells in Picture 6 are old and are -being destroyed;the haemoglobin has mostly gone, and all that isleft is the coat of the cell-its membrane or envelope.Partly destroyed red cells lie this are often seen in theurine.Patients who pass red cells in their urine are said tohave haematuria. If there is enough blood in the urine itwill be turbid, ‘smoky’, or even obviously red. Red cellsare sometimes destroyed in the circulation, and haemoglobinrather than red cells may be passed in the urine.The patient is then said to have haemoglobmuria. Hisurine will be red and clear, and there will be no red cellsto be seen in the spun deposit. Sometimes there are bothhaemoglobm and red cells in the urine.Pus cells are drawn in Pictures 7 and 8. In Picture 7they are fresh, and the nuclei can be seen quite easily.Pus cells are polymorph leucocytes which have comefrom the blood. They have a nucleus with several segments(see Picture G, FIGURE 7-9). These segments canoften be seen with a high power objective. As pus cellsare broken down and destroyed in the urine, their nucleidisappear and only the cell membranes remain. This hashappened in Picture 8.Epithelial cells are shown in Picture 9. These are largeflat cells with a nucleus that can usually be seen quiteeasily. They come from the epithelium or ‘skin’ on theinside of the ureters, bladder, and urethra. Epithelial cellsare usually single-that is, there is only one cell at atime. But groups of several epithelial cells are sometimesseen. A group of several epithelial cells is shown inPicture 9a and a very large epithelial cell with two nucleiin Picture 9b. Sometimes epithelial cells are more or lessegg-shaped, as in Picture 9c. A few epithelial cells arefound in normal urine. In this they are unlike red cellsand pus cells, which are abnormal and only found inurine when there is some disease of the urinary tract.When urine is formed in the kidney all the chemicalsin it are in solution. But, after it has left the kidney, someof these chemicals may become solid and form a urinarydeposit. Sometimes they form small solid granuleswithout any special shape, as in Picture 12. Deposits ofthis kind are said to be amorphous (without shape).These deposits are very common. Often, however, thesechemicals, as they become solid, take on the shape ofcrystals. When seen from on top the crystals in Picture13 are square with a cross on them like the envelope inwhich you post a letter. These are oxalate crystals. Otherkinds of crystal are shown in Pictures 14, 15, 16, and 17.But there are many other shapes also which have not beendrawn here. The shape of the crystals often tells us whatthey are made of. But crystals in the urine seldom matter.They will not therefore be described any more here.Picture 18 shows you what spermatozoa look like.Spermatozoa are the male sex cells which join with theegg inside a woman. From their joining a baby grows.Spermatozoa are often found in a man’s urine and arequite normal. After sexual intercourse they may be foundin a woman’s urine.Picture 19 shows you Trichomonas vaginalis. Thisprotozoon is described in Section 11.8. It is often seen inthe urine and gets into the urine from vaginal discharges.If you see a moving protozoon in the urine, it is almostcertainly T. vaginalis.Many micro-organisms are seen in the urine. Bacteriaare common. Like all the very small things in this figure,bacteria are only seen well with a high power or oilimmersion objective. Many bacteria are motile-that is,they move. The different kinds of motility are veryimportant and are described in the next section.Bacteria are very important but on@ in fresh urinethisis in urine which has been passed by the patient notmore an hour before,. or which has been in a refrigeratorfor nr,L more than 6 hours. Bacteria often grow in normalurine if it is left in a warm room. They get into the urinefrom a dirty jar, or from the air, or from the patient’sskin. Finding bacteria in old urine therefore means nothing.But, when bacteria are found in fresh urine, thismeans that the patient has a urinary infection. Whenbacteria are seen in fresh or refrigerated urine, they mustbe reported. Usually there will be pus cells and proteinpresent also.A common kind of bacteria found in the urine is calledEscherichia coli. It is a thin motile rod like a very smallpencil. The bacteria drawn in Picture 20 might beEscherichia coli.Other kinds of micro-organisms can often be seen inurine that has been left to stand in a warm place. Thebacteria in Picture 2 1 are growing in a group (or colony)of many hundreds together. Another group of bacteriahas been drawn in Picture 24. In this picture the placewhere the end of one bacillus joins the end of another hasbeen left as a space, but they are really stuck together.The threads or mycelium of a mould or fungus havebeen drawn in Picture 22 (see Section 11.15). The myceliumof moulds are longer and thicker than a bacterium,and they branch, as in Picture 22a. Some yeasts havebeen drawn in Picture 23. When yeasts grow, a parentcell ‘a’ often makes a daughter cell ‘b’, which is sr.Jerand grows from one end. Some fungi can grow either asyeasts or as moulds. In Picture 23c two yeast cells havebeen drawn which look as if they are going to grow in theform of a mould.Pictures 25, 26, and 27 in FIGURE 8-10 show some ofthe things which may muddle you if you do not knowwhat they are. Picture 25 shows a small bubble of air,and around it is the urine with an epithelial cell ‘a’, a puscell ‘b’, and an oxalate crystal ‘c’. Inside the air bubbleon the surface of the slide are some small bits of debris(dirt) ‘d’. Picture 26 is a piece of cotton thread that hasgot into the urine. Picture 27 shows two scratches onthe slide.

Three kinds of movement 1 8.148.14 Three kinds of movement (FIGURE 8- 11)You will see three kinds of movement in a wet film ofurine, stool, or blood. The first kind of movement is themovement of living micro-organisms themselves. Weshall call it motility (movement). Motile bacteria in theurine, like Escherichia co,li. can be seen swimming fromone place in the field to another, as in Picture 1, whileeverything else stays still. Some other micro-organisms,such as trypanosomes, microfilariae, and amoebae, havetheir own special kinds of motility. Microfilariae andtrypanosomes move very fast. Amoebae move veryslowly (see Section 10.7).The second kind of movement is called Brownianmovement. If you look at any very small particle lyingfree in a liquid, you will see that it is always moving andMO-n LlTY BROWNIAN STREAMINGMOVEMENTfl/-in:-. all the red cells in1 iiacterium r very small2 particlelacterium a SMALL particle isnoving from one part showing VERY SMALLthe field to another movements in-any directionTHESE ARE THREE MICROSCOPIC FIELDSFig. 8-l 1 Three kinds of movementmicroscope$bseen under theis never quite still. These movements are very small andquite fast. They are in any direction (towards any place),and they cti only just be seen. A small particle in themiddle of Picture 2 is showing Brownian movement.Because the movements are very small and in any direction,the particle does not move from one place toanother, but only shakes about.The third kind of movement we will call streaming. Ifthe coverslip over a wet film has not been properlysealed, the liquid dries up at the edges. This makes liquidmove or stream out from the middle of the coverslip tothe edges. In this streaming movement you will see astream or river of particles moving in the same direction,and the rest of the field may be still. At other timeseverything in the field will be moving towards the sameplace. Streaming makes it difficult to look at things in afilm. Some red cells are streaming across the field inPicture 3. Stop films streaming by sealing the edges ofthe wet film with Vaseline and paraffin wax as shown inFIGURE 7- 14.8.15 Looking for the ova of Schistosoma haematobiumIn some parts of the world one of the most importantthings to be found in the spun deposit of the urine are theova of a worm which lives in the veins around the blad-der. This worm is called Schistosoma haematobium. Itlays eggs that go through the side of the bladder into theurine. The disease that it causes is called scI&tosomiaslsor bilbarziasis. With the ova in the urine there areusually also protein, red cells, and pus cells. Most puscells are neutrophil polymorph leucocytes that havecome from the blood. In schistosomiasis many of the puscells may be eosinophils.The ova of Schistosoma haematobium have beendrawn in the first four pictures of FIGURE 8-10. InPictures 1 and 3 the embryo (the little schistosome insidethe egg-shell) is alive and is partly grown. It will soon beready to leave the egg-shell and swim free in the water. InPictures 2 and 4 the embryos are dead, and the egg-shellscontain granules of different sizes. In Picture 4 theremains of the embryo have cbme away from the shell ateach end. Notice that S. haematobium has a spine at theend of its shell-a terminal or end spine. In this it differsfrom S. mansoni. S. mansoni is almost always found inthe stool and has a spine on one side (a lateral or sidespine- look at Picture 5, FIGURE 10-7). The ova of S.mansoni with their spines at the side are sometimes seenin the urine, but this is rare.When you are looking for tile ova of S. haematobiumyou should take a specimen that has been passed by thepatient between midday and two in the afternoon. It is inurine taken at this time that the ova of S. haematobiumare most likely to be found. If it is not convenient to takean afternoon specimen, take any specimen and examinethat. If you find the ova, well and good. If you don’t, lookat an afternoon specimen. There are more ova in thebeginning and end parts of a specimen; so always takethe first and last parts of a specimen whenever you lookfor the ova of S. haematobium.The complete method is shown in FIGURE 8- 12. Veryoften 10 ml of a well-shaken 2 p.m. specimen will showthe ova, and the method can start at Picture 9, FIGURES- 12. In this diagram the deposit has been drawn as if itwas large and easily seen. This makes the diagramclearer, but when doing the method there is likely to bemuch less deposit than is drawn here. Even if no depositcan be seen, follow the method just the same, and do allthe steps just as if you could see a deposit. Schistosomeova are very small, and even if you can see no depositwith your eye you may still be able to find ova with amicroscope.METHODEXAMINING THE URINE FOR THE OVA OF s. HAEMATOBIUM.FIGURE 8-121. Ask the patient to pass his urine into a large bottleat two o’clock in the afternoon.2. Shake the bottle well.3. Fill a specimen glass with it.4. Let the specimen glass stand for at least 30minutes.5. At the end of this time any schistosome ova willhave fallen to the bottom of the specimen glass.

pour the urine intoa deposit may haveformed, this willcontain the ova030Mthis is the time ,:: ‘,of day when you I’- ‘1 --,- ;_ ii’should take theL-Jspecimen of urinetake as much urineas you canshake the bottlespin the urine for10 five minutestake away the rest. pour the urine intothere will prol+bly this is the last 10 ml -I a centrifuge tubebe a depositremainingpour away almost all thesupernatantthere will probablybe a depositmix up the deposit in‘+e urine with a deposit LOW POWER OBJECTIVE UNTILPasteur pipetteOVA ARE SEENFig. 8-l 2 Examining the urine for Schistosoma haematobium

Looking for the ova of Schistosoma haematobium 1 8.156. Without disturbing the deposit, carefully pour awaythe supematant urine so that only 10 ml is left (71.8. Wiih a Pasteur pipette carefully mix the depositwith the last 10 ml of urine.9. Pour this urine, which has the deposit mixed inwith it, into a lo-ml centrifuge tube.10. Centrifuge for at least 2 minutes. An electriccentrifuge is shown in this diagram. but a hand one willdo very well.11. A deposit will usually be seen at the bottom of thetube.12. Pour off all but the last 2 ml of urine (13).14. With a Pasteur pipette remove and throw away allbut the last drop of urine above the deposit (15).16. Mix the deposit in the last drop of urine, and takeit all into a Pasteur pipette.17. Place the last drop of urine with the deposit on aslide. Cover it with a coverslip. Using the low power ofthe microscope, search the whole slide until ova arefound. Follow the method shown in Figure 6-14.Count and report the number of ova you see. Reportalso how much urine you looked at. Reports might readlike this:‘5 ova of S. haematobium found in a deposit from10 ml of urine.’‘About 50 ova of S. haemafobium found in thedeposit from about 100 ml of urine.’‘Over 1,’ A ova of S. haematobium found in thedeposit from 200 ml of uritie.’In countries where S. haematobium is found, look fortheir ova in the urine of any patient who says he ispassing blood in Lis urine, or who has proteinuria,dysuria, or frequency.QUESTIONS1. What is a pus cell? Where might you find them?What do they look like?2. What different kinds of movement can you seewhen you look at a wet film with a microscope?3. What is a cast? How would you look, for them, andwhat does it mean if you find them?4. Why do we test the urine for sugar? What doesthe finding of much sugar and acetone in the urinemean?5. List six things that might be found in a urinarydeposit. What do they mean?6. What is PAS? Why and how do we test the urinefor it?7. What is bilirubin? What is it formed from? Whereis it found? How may it leave the body?8. What does it mean to find protein in the urine?9. How can urinary schistosomiasis be diagnosed?10. What micro-organisms are found in the urine?

9 1 The Cerebrospinal FluidLumbar puncture is usually done only on seriously illpatients. If the wrong reports are sent the wrong treatmentmay be given and the patient may die. Specimens ofCSF are therefore some of the most important specimensever sent to a medical laboratory.THE CSF AND HOW IT IS OBTAINED-SOMEHELP FOR MEDICAL ASSISTANTS9.1 Where the cerebrospinal fluid comes fromtThe spinal cord is a big nerve which joins on to thebottom of the brain. It is about as thick as the end ofyour little finger and goes down the back inside the manybones (vertebrae) which form the spine. The spine is alsocalled the vertebral column or backbone. The brain andspinal cord are soft and e&y hurt and are protected bybeing covered with several coverings or membranes.These coverings are called the meninges. When they areinfected by micro-organisms the patient is said to havemeningitis. Close around the brain and spinal cord is athin membrane called the pia mater. Around this is aloose membrane rather like a spider’s web. This is calledthe arachnoid mater. Outside this is a thick membranecalled the dura mater. This lies close inside the bones ofthe skull and the spine.We have said that the arachnoid mater is a loosemembrane like a spider’s web. From this you might thinkthat there would be air between the threads of the web.Instead of air there is a clear, saline solution called thecerebrospinal fluid or CSF. The CSF is everywhere betweenthe threads of the arachnoid mater and washes allround the brain and the spinal cord. The space filled withCSF is called the subarachoid space. When the brain orthe membranes covering it are diseased, it is useful to take aspecimen of CSF from the patient and look at it. Thiscan be done by putting a needle into the subarachnoidspace and letting some CSF come out. The needle is putFootnote. The first few sections of this chapter on the diagnosis ofmeningitis have been written in the belief that meningitis is too oftenmissed and that lumbar puncture is not done nearly often enough orwell enough. It is in line with other chapters which describe howspecimens are obtained, as well as how they are examined, and isintended primarily for those parts of the world where health centres arerun by medical assistants.into the lower part of the back or lumbar region, andputting it in is called lumbar puncture.FIGURE 9- 1 will explain all this more clearly. On theleft, in Picture A, is the skull (the bones of the head) andunderneath it is the spine. In Picture B the skull and thespine have been cut down the middle. The brain can beseen inside the skull and the spinal cord inside the spine.Between the brain and the skull, and between the spinalcord and the spine, you will see the subarachnoid spacefilled with CSF. You will see that the spinal cord endsbefore it reaches the bottom of the subarachnoid space.In Picture B the lower part of the subarachnoid space hasbeen drawn as if it were nearly empty, but in a livingperson it is full of spinal nerves which leave the spinalcord through holes between the vertebrae. i”o make thepicture simpler only one of these nerves has been drawn.If a needle is put into a patient’s back between thevertebrae and pushed into the subarachnoid space, theCSF will tlow out. Such a needle has been drawn inPicture B. When the spine is straight the vertebrae areclose together, and it is dimcult to put a needle into thesubarachnoid space. The patient’s spine must thus bebent if the needle is to get into the subarachnoid space.This is easier if the patient lies on his side. In Picture Csuch a patient has been drawn lying on his side with hisback bent and his knees close up under his chin. There isa needle in his back, and CSF can be seen dripping out ofit. In Picture D such a patient has been cut in half. Theneedle can be seen going into the subarachnoid space inbetween the spinal nerves.The most common reason for doing a lumbar punctureis to see if a patient has meningitis.9.2 The importance of lumbar punctureMeningitis in babies and young children is different frommeningitis in older children and adults. Babies andyoung children get meningitis more often. They getdifferent kinds of meningitis. They are affected differentlyby it and are diagnosed in different ways. Babiesare also more easily lumbar punctured. Much of whatyou will read in the next sections depends upon thesedifSerences.

Diagnosing meningitis 1 9.3IF THE PERSON IN PICTURE CWERE CUT IN HALF WHERE THENEEDLE IS, THIS IS WHATYOU WOULD SEEsub-arachnoidfull of CSFspaceDsub-arachnoidfull of CSF-body of II. vertebraeSPINALCORD2Isub-arachnoid soace -4fullofCSF (Q-intervertebral 8disc3-chest reeion(thoracicregion)PTHIS IS SOMEONE HAVING ALUMBAR PUNCTUREI“-535lumbar regionthis is justone of themany spinalnerves thatleave thespine betweeneach vertebralboneJ\ this space is fullof spinal nerves,only one is drawnFIRM BED : PAT lENiNEAR THE EDGE OF THE BED :THE PATIENT’S BACK IS WELL ARCHED : THE NEEDLEIS GOING STRAIGHT IN : VERY CAREFUL ASEPTICPRECAUTIONS ARE BEING OBSERVEDFig. 9-lWhere the CSF comes fromIf a patient is thought to have meningitis the first partof the diagnosis is to make sure that he really has gotmeningitis and not some other disease. The second partof the diagnosis is finding out what kind of meningitis hehas. In adults and older children the history and examinationof the patient will usually tell us if a patient hasmeningitis, even without a lumbar puncture. In babiesand young children we have often to do a lumbar puncturebefore we can tell that they have meningitis and notsome other disease. After meningitis has been diagnosedwe must know what kind of meningitis it is. For this,lumbar puncture is always necessary.Meningitis is easily diagnosed in adults and older children.Such patients are not common and can usually beadmitted (taken) into a hospital ward for lumbar puncture.But there are almost always too many young childrenand babies for all those who might have meningitisto be admitted to a ward. If meningitis is to be diagnosedthese children must therefore be lumbar punctured inhealth centres and outpatient departments. Medicalassistants in health ceritres must thus be taught how todiagnose meningitis by lumbar puncture. This means thathealth centres must have the right equipment, both forthe puncture itself and for looking at the CSF afterwards.It is also useful to be able to examine the CSE’ in anoutpatient department.9.3 Diagnosing meningitis (FIGURE 9-2)It is usually easy to tell if an adult or older child hasmeningitis. He looks ill, he lies with his head back andhis eyes shut, and he does not want to be moved. If he isold enough, he may tell you he has a headache. But ababy or young child may show none of these things. Allhe may show is fever (for which there is no other cause),fits, abnormal sleepiness, or, if he is very young indeed,

A HEALTHYheadPERSONthe head of a healthyperson can be bentforward so that his chindiagnosistraighteied when his legsA PATIENT WITH MENINGITISD a patient with meningitiscannot touch his chestEa patient with meningitis cannotbe made to straighten his knee F _a patient with meningitis cannotwhen his leg is raised without -““v put his head between his knees(mandible).posterior(back)when a child hasequipment:.:. :.;.*L, :_.‘:‘i :-: .., :< ._ #.-.,rte ;::.:,:..:..,;;‘;,.;,”.,.. I. .::.....-i,.,, _‘.,.Z I..:.: ‘...;*:::,.:I..anterior (front) :‘:“I’fytanelle . .,‘..‘.?.:AJSPECIAL LUMBAR PUNCT’JRE NEEDLEKtoLrt$r this partthe*r&dle_I IFig. 9-2The signs of meningitis

Equipment for lum’bar puncture 1 9.4he may just stop suckling. These are the children whoneed lumbar puncture.There are three useful ways of examining an adult orolder child to see if he has meningitis. In the first way liethe patient on his back. Put your hand behind his headand lift it so that the patient’s chin Louches his chest. Youwill find that you cannot do this because the patient’sneck feels stiff and hurts when it is moved (Picture D).The chin of a normal person or of a patient who has notgot meningitis can easily be made to touch his chest(Picture A). Most older patients with meningitis have this‘neck stiffness’.The second way is a similar one that you can do withthe legs of patients who you think might have meningitis.If you lie a normal person on his back, and bend one ofhis legs forward over his abdomen (stomach), you willstill be able to straighten his knee quite easily (Picture B).If, however, the patient has meningitis you will be unableto straighten his knee when his leg is bent forward overhis abdomen. If you try, the muscles at the back of his legwill feel sti5 and will hurt as they are pulled on (PictureE). When this happens the patient is said to show apositive Kernig’s sign and is likely to have meningitis.The third method is to try to get a patient’s headbetween his knees. If the patient has not got meningitisyou will probably be able to do this quite easily (PictureC). If the patient has meningitis you will be unable to gethis head between his knees, because the meningitis willmake his back too stiff (Picture F).Young children and babies may show none of thesesigns. They have, however, a special sign of their own.This is the ‘bulging fontanelle’. Bulging’ means swellingor sticking out. The fontanelles are the soft places betweenthe bones of a baby’s skull and are shown inPictures G and H. The fontanelles bulge because thepressure inside the skull is increased. Meningitis is themost important cause of an increased pressure inside theskull. The fontanelles close up as a child grows older; soa bulging fontanelle is not seen in older children andadults. To find this sign put your hand on the baby’s headand feel for these fontanelles. Are they normal or arethey bulging?9.4 Equipment for lumbar punctureIn adults and older children a special lumbar punctureneedle is necessary. This is longer and stronger than theneedles that are used for giving injections and has a styletinside it. A stylet is a wire that goes down inside theneedle and closes its end. This needle and its stylet areshown in Pictures K and L, FIGURE 9-2. When theneedle is in the subarachnoid space, CSF will only comeo&t when the stylet has been removed.In babies a thin sharp intramuscular needle can beused for lumbar puncture, but it is better to use a specialsmall lumbar puncture needle and stylet. Intramuscularmeans ‘between the muscles’ and is the name given to thekind of needle that is used for giving injections. A styletis not needed.A LUMBAR PUNCTURE NEEDLE MUST BESTERILE. If the needle is not sterile, or if microorganismsget on to it from your hands while lumbarpuncture is being done, the patient may get meningitisfrom the needle. If you are not careful the microorganismson the needle may infect the patient’smeninges. Lumbar puncture can therefore be verydangerous and MUST be done with very good asepticprecautions like those described in Section 1.22. Thereare two ways of sterilizing lumbar puncture needles.They can be sterilized in boiling water, or they canbe autoclaved in a pressure cooker (see Section 1.21.).The equipment for lumbar puncture can also beprepared in two ways. The complete equipment can besterilized in a cloth roll with pockets like that in PictureI, FIGURE 9-2, and kept sterile until it is needed. This isoften very useful. In a young child lumbar puncture canalso be done with nothing more than a sterile intramuscularneedle. We will describe both ways, but, beforedescribing them, here is one very important word ofwarning.Before you first do a lumbar puncture, you must asksomeone who knows how to do it to show you. Watchhow he does it. Then ask him to watch you doing alumbar puncture on at least one adult and one child.After thai you may be able to do a lumbar puncture withonly an assistant to hold the patient.Until you have done many lumbar punctures, alwaysread the method again before you start.METHODPREPARING A SET OF LUMBAR PUNCTURE EQUIPMENT.PlCTURES I and J, FIGURE 9-2Prepare a cloth roll like that shown in Picture I. Makepockets for two lumbar puncture needles, two thin intramuscularneedles, some swabs, a syringe, and two bijoubottles for the CSF.Find a tin with a lid that will both hold the roll and gointo the pressure cooker. Put a small bowl or jar (a‘gallipot’) into the tin. Put the roll of equipment on top ofit. Find a theatre towel and cut a round hole or windowin the middle of it about 10 cm across. This is to putacross the back when you do the lumbar puncture. Foldthis towel and put it on top of the roll of equipment. Ontop of this put a small towel to dry your hands. Put thethings in the tin in this order, because this is the order inwhich you will want them.Put the tin with the equipment and towels on its sidein the cooker. This will allow the air to be driven away bythe steam more easily-see Section 1.21. Put the lidbeside the tin but not on it.Autoclave the tin at 15 lb for 15 minutes.Take out the tin without touching anything inside itand immediately shut its lid. Put a piece of adhesivetape across the edge of the lid. Write the word ‘Sterile’on it and the date. This roll can be kept until it is wanted.It should be sterilized each month, even if it has notbeen used.

9 1 The Cerebrospirw ’9.5 Doing a lumbar punctureNow that you have prepared the equipment you can do alumbar puncture.METHODDOING A LUMBAR PUNCTURE. PICTURE C, FIGURE 9-lLay the patient on his side on the edge of the bed. Aska nurse to put one hand behind his head and the otherhand behind his knees. Ask her to bend his back asmuch as possible so as to open up the spaces betweenthe vertebrae.Swab the lower part of the back (the lumbar region)three times with iodine, using a fresh piece of gauzeeach time. Use tincture of iodine, which is a solution ofiodine in spirit, not Lugol’s iodine.Open the lid of thetin. butdon’ttouch anything inside it.Scrub your hands well with soap and water, if possibleunder a running tap. From now on your hands aresterile and must not touch anything unsterile until lumbarpuncture is finished. Dry your hands on the steriletowel in the tin.Put the second sterile towel with the ‘window’ in itacross the patient’s back, so that the window is over thelower part of the back where you want to put in theneedle.Feel for the iliac crest through the towel. The iliaccrest is the top part of the pelvic bone. Follow a linefrom the iliac crest down across the back. This line isshown in Picture C. This will cross the fourth lumbarspine or the space between the, third and fourth spines.You can easily feel the lumbar spines and the spacesbetween them through the skin. You can put your needleinto any space provided it is below the second spine.Wet the ends of your fingers with iodine and keepthem wet while you touch the needles.Inject about 1 ‘ml of local anaesthetic, such as 2%lignocaine or procaine solution into the skin in themiddle of the space where you want to put the lumbarpuncture needle. If possible, take the local anaestheticfrom an ampoule, not from a rubber-capped bottle.Take the lumbar puncture needle and push it betweentwo spines through the place where you have injectedthe local anaesthetic. Push the lumbar puncture needlestraight forwards. Some people try to point it at theumbilicus. You will feel the needle suddenly go in moreeasily as it goes into the subarachnoid space. GATake out the stylet. CSF should come out of theneedle. If no CSF comes out, turn the needle round, putback the stylet, and take it out again. If still no CSFcomes, ask the patient to strain so as to raise his CSFpressure.If the needle hits bone, pull it out a little, make sure itsdirection is right, and push it in again. If you still cannotget any CSF try the next space.As soon as CSF flows, put about 5 ml into each of thetwo bottles.If the child is under 3 years you can do a lumbarpuncture like this.METHODLUMBAR PUNCTURE WITH AN INTRAMUSCULAR NEEDLEChoose a long, thin, sharp intramuscular needle. Boilit together with a small bowl for at least 5 minutes infast-boiling water. Lift the bowl out of the boiling waterwith a pair of sterile forceps. Lift the needle out of theboiling water with the sterile forceps and put it in thebowl. Touch only the adaptor with the forceps.Wash and have the child held as described above.Hold the intramusc lar needle by the adaptor and/push it into the space between the vertebrae. It will goin very easily. You will need only 2 or 3 ml of CSF.Watch these points very carefully. If you do not watchthem carefully, you may give your patient meningitis byputting the micro-organisms on your hands or a dirtyneedle into his CSF.KEEP THE NEEDLE STERILE. Never touch anypart of a needle except the adaptor with your fingers.Never put the needle anywhere unsterile before you put itinto the patient. If you are using a sterile roll of equipment,put little glass tubes or test tubes in the pockets ofthe roll for the needles. If you want to put a needle downbefore putting it into the patient again, put it into one ofthese sterile tubes.Dip your fingers into iodine before you touch theneedle. If micro-organisms remain on your Angers afteryou have washed them, this will help to kill them. Somepeople like to use spirit or an antiseptic called Cetrimide,but iodine is a better antiseptic.If you can, wear sterile surgical gloves and a surgeon’smask.If you are putting a needle into a patient’s CSF, take itstraight out of boiling water. Don’t let it stay at thebottom of a wet dish where it may become infected againbefore you use it. A freshly boiled needle is safer thanone which has been badly autoclaved or stored too long.If lumbar puncture is going to be easy, the patientmust be held properly. His back must be bent as much aspossible and must be right on the edge of the bed. The bedor bench should be firm andflat.Put the needle into the middle of the space between thevertebral spines. Put the needle in straight. It must not besloping, either upwards or downwards or towards thelegs. Some people point the needle very slightly towardsthe head as they put it in.Each lumbar puncture needle has its own stylet. Don’tmix them up.HOW THE CSF IS EXAMINED9.6 Two specimens of CSFTwo specimens of CSF should be sent to the laboratoryfor each patient. Specimen One is the first few millilitres

Cells 1 9.9of CSF to flow out of the lumbar puncture needle. SpecimenTwo is the CSF that comes next. Sometimes adoctor or medical assistant has difficulty in getting hislumbar puncture needle between the vertebrae into thesubarachnoid space. He may have to try several times,and the patient may bleed a little from the needle holeinto the CSF. This is often called a ‘bloody tap’. Whenit happens the first specimen of CSF will probably containmost of the blood. and the second specimen maycontain little or none. If there are two specimens, try todo as many tests as possible on the second one.Clear or turbid?The first +&ing to do with a specimen of CSF is to look atit. Normal CSF is perfectly clear-like pure water.When there are about a hundred cells per cu mm in theCSF it will look very slightly turbid. Hold the specimenup to the light-if it is not clear like water it is likely tocontain at least a hundred cells per cu mm.ColourNext look at the colour of the CSF-some specimens ofCSF are yellow. If there is blood in the CSF the yellownessmay only be seen in the supematant fluid after thespecimen has been centrifuged. Yellowness of the CSF iscalled xaathoehromia.9.9 Cells (Normal, less than 5 per cu mm)White cells are looked at in two ways. We count thenumber of cells per cu mm of CSF. If necessary, we alsospread the cells in the centrifuged deposit on a slide andstain them with Gram’s stain or Leishman’s stain.Cells in the CSF are counted in the same way as cellsin the blood, but, because there are fewer cells in theCSF, it is best to use a deeper chamber which will containmore CSF. A Fuchs-Rosenthal chamber O-2 mm(+ mm) deep is therefore better than a Neubauer chamber.which is only O-1 mm (&, mm) deep. But a Neubauerchamber can, if necessary, be used for both blood andCSF. So as to save money it is the only chamber in themain list of equipment. There is, however, a Fuchs-Rosenthal chamber in Choice 5, Section 13.18. Thesechambers are described very carefully in Section 7.29.Ways of using both chambers for CSF are described inFIGURE 9-3. This shows drawings of the three differentkinds of counting chamber that you may have. You willsee that in each chamber there are several blocks ofsixteen small squares.When there are many cells in the CSFIf there are many cells, say more than fifty in each cumm, it will be enough to count the cells in one block ofsixteen small squares. With the Neubauer chamber, multiplythe number you find by ten. With the Fuchs-Rosenthal chamber, multiply the number you find by fivebecause it is twice as deep. This is shown in Pictures Aand B, FIGURE 9-3.Whenthere are few cells in the CSFIf there are a few cells in the CSF, a larger area of thechamber must be counted. This is especially important ifthere are about five cells per cu mm because this is thenumber that divides the normal from the abnormal.If there are few cells in the CSF, and you are using theNeubauer chamber, count the cells in the whole ruledarea and then count one more block of sixteen smallsquares a second time (it does not matter which). Thiswillbe9x0~1cumm+0~1cumm=l~0cumm.Thisway of counting is shown in Picture C. Provided theCSF has not been diluted, this will be the number of cellsin 1 cu mm and no multiplying will be needed.It is because counting the whole ruled area of a shallowNeubauer chamber takes so long that the deepFuchs-Rosenthal chamber is used. With this chamberyou need only count five blocks of sixteen small squares.This is 5 x 0.2 = 1.0 cu mm. It is shown in Picture D.Provided that the CSF has not been diluted no multiplyingis needed. The ruled area in the modified (altered)Fuchs-Rosenthal chamber (Picture D) is smaller thanthat in the standard Fuchs-Rosenthal chamber (PictureE). The modified chamber is the one in the main list ofequipment.The cells in the CSF can be counted by putting CSFstraight into a chamber undiluted and unstained. Thismay be the best thing to do if one single-celled chamberis all you have. But, if you have a double-celled chamber(or can use two single-celled chambers), it is best todilute the CSF with an equal volume of white cell dilutingfluid. This is the same fluid that is used to count thewhite cells in blood (see Section 7.29). This fluid destroys(haemolyses) any red cells present and stains thewhite cells blue. Staining makes the white cells easier tosee. If you are looking at plain CSF in a single chamberand you find it dimcult to decide what kind of cells youare seeing, it may help if you clean the chamber and lookat more CSF, this time diluted with white cell dilutingfluid. If CSF has been diluted with an equal volume ofwhite cell fluid, the number of cells counted must bemultiplied by two to make up for this dilution.It is important to know what kind of white cells thereare in the CSF. Are they polymorphs or lymphocytes? Itis usually easy to see which they are by looking at themcarefully in a counting chamber with a high power objective.If the CSF has been diluted with white cell dilutingfluid, you will easily see the nuclei of the white cells. Thenucleus of a polymorph has several segments. The nucleusof a lymphocyte is round or nearly round. Alwayslook at white cells in a counting chamber carefully in thisway. You can also tell if the cells are polymorphs orlymphocytes by looking at them in a Gram-stained film(a Leishman film is better), but this is not usually necessary.The main use of a Gram film is to stain bacteria.In normal blood there are about 5,000,OOO (five mil-

p,.:- ,i ‘..,_z:$-_ 9 1 The Cerebrospinal FluidA NEUBAUER RUL!NG B MODIFIED FUCHS-ROSENTHAL RULING\many cellsfor undiluted CSFcount the cells in16 small squares andmultiply it by 10for undiluted CSFcount the cells in16 small squares andmultiply it by 5C NEUBAUERRULINGD MODIFIED FUCHS-ROSENTHAL RULINGfew cellsE STANDARD FUCHS-ROSENTHAL RULING‘for undiluted CSFcount the cells inall parts of theruled area andin one extra block of16 small squares: this will be onecubic mm and thereis no need to doany multiplyingF MICROSCOPIC VIEW THROUGHTHE HIGH POWER OBJECTIVEfor undiluted CSFcount the cells infive blocks of 16small squares : thisis one cubic mm andthere is no need todo any multiplyingthese dotted linesare in the diagramonly : they are notruled on the chambersome Fuchs-Rosenthalchambers are made likethis : five blocks of16 small squares areone cubic mm.-ruledlineslymphocyteremember to multiply your answer by two if you havediluted the CSF with white cell diluting fluidFig. 9-3 Counting cells in the CSFlion) red cells and 5,000 (five thousand) white cells ineach cu mm of blood. If blood from a ‘bloody tap’ getsinto the CSF there will therefore be about 1,000 red cellsfor every white cell. In a ‘bloody tap’ the CSF proteinwill also rise about 1 mg for every 1,000 red cells. It isuseful to know this, because you may be sent a specimenof CSF which has some blood in it. The person lookingafter the patient will want to know if all the white cellsand protein in the CSF specimen are due to the b!ood, orif there were white cells and protein in the CSF beforethe blood got in. You will be able to tell him by countingthe red cells. Allow one white cell for every 1,000 redcells and 1 mg of protein for every 1,000 red cells. Bydoing this it is usually easy to tell if the white cells and4

Stained films 1 9.11protein that you find in a specimen of CSF have beencaused by a ‘bloody tap’. This is the only reason forcounting the red cells.9.10 Pandy’s method (Normal CSF protein less than35mg%)The CSF protein is examined in two ways.A rough measure of the CSF protein can be found veryeasily by using Pandy’s method. One or two drops of CSFare allowed to fall into a little tube of Pandy’s reagent(see Section 3.37), which is a saturated solution of phenolin water. If there is less than 25 mg % of protein thereis never any turbidity. If there is more than 35 mg % ofprotein in the CSF, the mixture of protein and Pandy’sreagent always goes milky. When there are between 25 and35 mg % of protein the CSF mixture sometimes goesmilky and sometimes stays clear. When there is more than35 mg % of protein in the CSF it is abnormal. Pandy’sreagent is thus a very quick and useful method of tellingwhether the CSF protein is normal or abnormal. If CSF isabnormal in any way Pandy’s test is almostalwayspositive.The other way of measuring the CSF protein is moreaccurate. One ml of CSF is mixed with 3 ml of 3%sulphosalicylic acid. Protein in the CSF makes the mixtureturbid. The more protein there is, the more turbidwill the mixture become. This turbidity is measured witha set of proteinometer standards, or with an MRC Greywedge photometer, which is described in Section 9.13.9.11 Stained filmsIt is usual to count the cells and measure the protein inall specimens of CSF sent to a laboratory. Very often, afilm of the spun deposit is also stained by Gram’smethod. Gram’s method stains the bacteria in the CSFwhich cause bacterial meningitis. Some people also staina film of the spun deposit by Leishman’s method (seeSection 7.12). This makes it easier to see if cells arelymphocytes or polymorphs. Films can also be stainedby the Ziehl-Neelsen method (see Section 9.18).9.12 A combined method of examining the CSFThe combined method which follows and which is describedin FIGURE 9-4 may look difficuit because severalmethods are combined- -cells. Pandy, protein, film, andculture. It is worth following very carefully because itsaves much time. A combined method also makes thebest uqe of a small quantity of CSF. There is usuallyenough blood, stool, or urine in a specimen for all themethods we want to do. But there is never more thanabout 5 ml of CSF; so it has to be used very carefully.In some laboratories it is possible to culture theCSF-that is, to grow the bacteria in it. A method forculture has therefore been included. This will not bepossible in most of the hospitals and health centres wherethis book will be used, but the extra steps for culturingthe .CSF can easily be left out. In some of the steps in thefollowing method it is important not to get any organismsinto the CSF-that is, not to contaminate it. Thesesteps are marked sterile. In the method which follows theuse of a double-celled chamber is described. But it caneasily be altered for use with a single-celled chamber.METHODLOOKING AT THE CSF-A COMBINED METHOD, FIGURE S-4In a test tube rack put a sterile plugged cesrtrifugetube (Picture 5; an unsterile centrifuge tube can be usedif no culturing is being done), a graduated centrifugetube (Picture 241, a small test tube such as ML 48b(Picture 131. and a Kahn tube full of Pandy’s reagent(Picture 9). Put the coverslip on the counting chamber(Picture 14). If the CSF is to be cultured you will needtwo Pasteur pipettes-these have been marked ‘A’ and‘B’. If you are not going to culture the CSF you will onlyneed one Pasteur pipette.1. If you have been sent two bottles of CSF, do asmany- tests as you can on the second bottle.2. Mix the CSF well if it has stood for more than a fewminutes.3. If the CSF is to be cultured flame a Pasteur pipette.4. Using ‘aseptic precautions’ if the CSF is to be cultured(that is, taking care not to let any organisms thatmay be on your fingers or elsewhere get into the CSFI,put most of the CSF in the second specimen bottle intothe sterile plugged centrifuge tube (Picture 51. If a glasscentrifuge tube is being used it is wise to leave a littleCSF in the specimen bottle in case the tube breaks inthe centrifuge. If you have been sent two specimens ofCSF, the CSF in the first bottle can be looked at if thetube containing all the CSF from the second bottlebreaks in the centrifuge. Plastic centrifuge tubes do notbreak; so you will not have this trouble.6 and 7. If you have an electric centrifuge, put theplugged centrifuge tube on to spin while you get on withthe method as far as Picture 21. Besides having fi littleCSF in reserve (in case the tube in the centrifuge breakswhile spinning) you should have enough CSF left in yourpipette (81 for Pandy’s method (starting in Picture 9) andthe cell count (starting in Picture 13). Once you havefilled the plugged centrifuge tube there is no need toworry about aseptic precautions until you get to Picture23. When making a plugged centrifuge tube, make surethe plug is well made, or spinning will send it to thebottom of the tube.10. Put two drops of CSF into the tube of Pandy’sreagent. Normal CSF produces no turbidity (Picture 11 I.If there is an abnormal amount of protein in the CSF thePandy’s reagent will go turbid (Picture 12). Record theturbidity with the plus notation.13. Put two more drops of CSF into the small test tube.14. Fill one side of a double counting chamber withthe CSF remaining in the pipette.15. If there is any CSF left in the pipette, throw itaway into the lysol jar.

second specimenpipettsecond oftwo specimensif there ismore than onesteps3,4,5&6Bunsen burneradd t\to the r Pandy’s reagent, 11 1 Itsterile’ \i[.?fabout 4 mlthis tube goes intothe centrifuge inPOSITIVE,the mixturenail test:4.PANDY’STESTNEGATIVE,stays clearthe mixtureadd two drops of CSFa small test tubeadd two drops of white ceidiluting fluid to the CSFin the small tubesome white cclof CSF and whcell diluting fluidempty the pipettefill one side of a doubleFuchs-Rosenthal countingchamberyou have now finished with pipette Aput the s&rilepluggedcentrifuge tube from step 6into the centrifuge70--.>-.--=I=== ;=.:.:,.C{, /4 :i',>,i .. .,.'('.i.::‘,. .,.,.C."..'Qr. .' I..,ii: .'. , _,_.._._. ;"-.this is the secondsterile Pasteurpipetteplugthis is a graduated centrifugetube for the CSF protein : put1 ml of the supernatant CSFinto it@-deposit u u.depositthrow away any remaining CSF into L&job26 Ysupernatantremovedop to make a filmFig. 9-4 A method for examining the CSF

,(The CSF protein 1 9.1316 and 17. Take some white cell diluting fluid into thepipette.16. Add two drops of white cell diluting fluid to thetwo drops of CSF in the small test tube.19. Throw away any remaining white cell fluid intothe lysol jar.20. Mix the XF and the white cell diluting fluid in thesmall test tubdraw it in and out of the pipette. Them‘brture will be half CSF and half white cell diluting fluid.Any red cells there may be will be lysed, and the whitecells will be stained a pale blue.21. Fill the second half of the counting chamber withthe mixture of CSF and white cell fluid. If you are culturingthe CSF put aside pipette A. Count the cells accordingto the diagram in Figure 9-3. Sometimes it may beuseful to count the red cells also. For this, see Section9.9.While you have been doing all this, the CSF in theplugged tube that you put into the centrifuge in Picture7 will have been spinning. It may have a deposit (22)after spinning.23. Flame a second sterile Pasteur pipette, and, takingcare not to touch the pipette with anything unsterile,put 1 ml of the supernatant fluid into a graduated centrifugetube (Picture 24). This is used to measure the CSFprotein in the next method.25. If you are going to measure the CSF sugar by themethod in Section 7.42, keep some of the supernatantfor this. Throw away any remaining supernatant into thelysol jar so that only the deposit remains.26. Mix the deposit with the last remaining drop ofsupernatant.27. If the CSF is being cultured put a few drops of thedeposit on Petri dishes of blood agar and chocolate agarand spread them out.28. Put a drop or part of a drop of the deposit on twoslides-only one is shown here.29. Spread out the deposit with a loop to make a film.Flame the film and stain it by Gram’s method (seeSection 11.5). If necessary, stain another film byLeishman’s method (see Section 7.12).9.13 The CSF protein (FIGURE 9-5)As we saw in Section‘:9.10, protein in the CSF can bemeasured with the Grey wedge photometer, or with a setof proteinometer standards. These instruments only givean accurate answer if the CSF is clear to start with. Thismeans that if it is turbid with red cells or white cells tostart with, it mustjkst be cenrrl$ugedIn both methods, 1 ml of CSF is mixed with .q mi of3% sulphosalicylic acid. The collection of 1 ml of centrifugedCSF has already been described as part of thecombined method of looking at CSF. The graduated tubein which the mixing is done is shown in Pi-Lure 24 inFIGURE 9-4. The rest of this section describes how to usethis specimen to measure the CSF protein.When we described how haemoglobin is measured, wesaid that it is possible to do this with a row of tubes withdiffering amounts of haemoglobin in them (FIGURE 5-4).These tubes were called standards. It is also possibleto use a set of standards of milkiness or turbidity tomeasure the turbidity of a mixture of CSF and sulphosalicylicacid, and thus the amount of protein in theCSF. The higher the CSF protein, the greater will be theturbidity.Haemoglobin standards can be made as pieces ofcoloured glass in a Lovibond disc, but standards formeasuring protein are made as a row of test tubes whichhave been carefully sealed (corked or closed). Such a setis drawn in FIGURE 9-5. There are several things toremember when using it. One is to use the same kind oftube as that in which the standards are made. A tube of adifferent size will not give a good comparison and willgive the wrong answer. Try therefore not to lose thespecial tubes for doing this test that are supplied with theproteinometer. If you do happen to lose these tubes, use atube which is as nearly like them as possible, such as aKahn tube. It is also important to match the test solutionwith the standards in the rack while light is coming overyour shoulder as in Picture 8. This will make it easierto find which of the standards has the same turbidityas the test solution. Another important point is to wait5 minutes before comparing.The Grey wedge photometer was made for measuringthr haemoglobin in blood, but by a fortunate chance it isalso possible to use it to measure protein in the CSF. Themixture of 1 ml of CSF and 3 ml of 3% sulphosalicylicacid is poured into a cell, and, using the green No. 1filter, the wheel of the Grey wedge is turned until the twosides of the field of view through the eyepiece are equal.The figure in the window of the Grey wedge gives theCSF protein in mg % and no tables are needed to workout the answer.METHODMEASURING THE CSF PROTEIN, FIGURE 9-51. Using a Pasteur pipette fill a graduated centrifugetube to the l-ml mark with CSF. This has been done inPicture 24, Figure 9-4.2. Fill the tube to the 4-ml mark with 3% sulphosalicylicacid as prepared in Section 343b.3. Mix gently and wait for 5 minutes.4. Fill either a glass cell for the Grey wedge photometeror, as in Picture 5, one of the special tubes for the setof proteinometer standards. Make sure that the cell forthe Grey wedge is clean, and that its outer surface is dry.USING THE GREY WEDGE5. Put the cell with the turbid mixture of CSF and 3%sulphosalicylic acid into the right-hand place in the cellcompartment of the Grey wedge photometer.6. Put a cell filled with water in the left-hand place.See that the green No. 1 filter is screwed into the eyepiece.Turn the wheel of the Grey wedge until the two

‘: ,* .’I; ‘_rr,,>i,’ ;:-/,) .‘,9 1 the derebrospinal Fluid3% sulphosalicylic acidhalves of the field of view look exactly the same. Thenumber on the scale in the window will be the CSFprotein in mg %. Sometimes there will be so much proifyou have not got a grey tein in the CSF that the two halves of the field of viewwedge photometer use a cannot be matched. When this happens, dilute someset of proteinometerstandards : this ismore CSF, es described below.one of the tubesUSING THE SET OF PROTEINOMETER STANDARDSstandards numbered here /7. You will see that the set of proteinometer standardshas ten small tubes, each of which is equal to adifferent amount of protein in the CSF. The lowest tubeis equal to 10 mg % of protein, and the highest to 100mg %. Put your tube of CSF and sulphosalicylic acid inthe rack and see which of the standards is most like it. InFigure 9-5 it is shown in between the tubes containing70 and 90 mg % of protein. It was a little more turbidthan the tube with only 70 mg % and a little less turbidthan the tube with 80 mg %. It thus had about 75 mg %of protein.another way of measuring 1 ml of CSF and What should you report if the test solution is more turbid3 ml of suluhosalicvlic acid solution is /than the 100mg standard. When this happens all that isto use the-special pipettes that come withusually needed is a report which says ‘CSF protein morethe set and clip in behind the standardsthan 100 mg %‘. Another thing to do is to put only f mlof CSF in the graduated centrifuge tube, add + ml ofsaline and then add the usual 3 ml of 3% sulphosalicylicacid. It may well be possible to find a match, but theanswer found must be multiplied by two. Sometimesthere may be so much protein in the CSF that evenfurther dilution may be needed. Diluting the mixture ofCSF and sulphosalicylic acid when it is already turbidwill not give you an accurate answer.STANDARDSthe set of standards is keot inabox,seeFigureZ-&ML36compare the test with the standards with lightcoming over your shoulderoverFig. 9-5 Measuring the CSF protein9.14 TrypanosomesIn Section 7.36 we said that the best way of findingtrypanosomes in the blood is to look for moving organismsin fresh blood. The same is true of CSF. If you areasked to look for trypanosomes in CSF, look for themlike this.METHODTRYPANOSOMES IN THE CSFSpin some fresh CSF as shown in Pictures 5,6, and 7in Figure 9-4. Spin it for at least 5 minutes as fast as youcan.Take off all the supernatant as shown in Pictures 23,25, and 26, Figure 9-4. There may be very little deposit-only a tiny white piece at the bottom of the tube. Takecare not to disturb this small deposit when removing thesupernatant. Using a very fine (thin) Pasteur pipette (ifneed be, pull it out in a flame to make it finer) mix whatdeposit there may be in the smallest possible amount ofsupernatant CSF. Put all the deposit on a slide. Cover itwith a coverslip. Using the high power objective and thecondenser racked down a little, search the whole area of

i r: .,_‘.,i, I,>‘,i,.2c:,, y-,...,-. , ‘ ‘II ,I, ;,,Sugar in the CSF 1 9.15 ’PNEUMOCOCCAL MENINGITISthe coverslip until moving trypanosomes are seen.When searching, follow Figure 5-14 so that no part of reptococcus pneumoniaethe coverslip is missed.If you are in a country where trypanosomiasis is seen,it is especially important to e-x-amine evev specimen ofCSF plain and not diluted with white cell diluting fluid.This is because white cell diluting fluid kills trypanosomes,and you will not see them moving. A doctor maynot think that his patient might have trypanosomiasisand may not ask you to look especially for trypanosomes.If you look at undiluted CSF you may see themmoving in the counting chamber. But if you dilute theCSF with white cell diluting fluid, the trypanosomes will Plates 96 & 97be dead and not moving; so you may not see them.HAEMOPHILUS INFLUENZAEMENINGITIS9.15 Sugar in the CSFMeasure this in the same way as you measure the blood bacilli,of different shapesGram negative [pink staining)sugar (see Section 7.42), except that when you use theLovibond comparator take four times as much CSF anddivide ih~ ZSwei by four. This, iA 0.4 ml of CSF andnot 0~ 1 ml. When the Grey wedge or EEL calorimeterare used it is best to take only twice as much CSF-0.2ml and divide the answer by two. Greater volumes ofCSF are taken because there is less sugar in the CSFPlate 96than there is in the blood. CSF should be taken into affuoride bottle in the same way as blood (see Section 4.6).Diseases alter the CSF in several ways. Here are someof the diseases you will meet and the changes they cause. cocci, mostly in pairsABNORMALITIESOF THE CSF9.16 Suppurative or bacterial meningitisthese are allGram filmsSuppurative means pus-making, and the CSF from apatient with suppurative meningitis often looks like pus.It is often obviously turbid and may be green or yellow. Fig. 9-6 Three kinds of bacterial meningitisThere are usually over 500 cells per cu mm, but theremay be as few as 15 or 20 cells per cu mm. Most of the able to tell what the likely species of bacterium is bycells are usually polymorphs and the rest lymphocytes. seeing whether it is Gram positive or Gram negative, andSuppurative meningitis is caused by bacteria growing whether it is coccus or bacillus. All three species ofin the CSF and meninges. Many species of bacteria can bacteria cause the same kind of suppurative meningitis incause meningitis, but three species of bacteria cause most a patient, but it is important to tell which species iscases. These bacteria can usually be seen when a film of causing the meningitis because they are killed with differtheCSF is stained by Gram’s method. Because each ent drugs. Films from each of the three types of meninspecieslooks different in a Gram-stained film, it is gitis are drawn in FIGURE 9-6. You will see that, evenusually easy to tell which of the three is causing the though Streptococcus pneumoniae and Neisseriameningitis. The three species of bacteria are meningitidis are both cocci, they are not quite the sameStreptococcuq pneumoniae (the ‘pneumococcus’), shape. Neisseria meningitidis is bean-shaped with the flatNeisseria meningitidis (the ‘meningococcus’), and sides of the bean together, but Streptococcus pneumoniasHaemophilus influenzae. We shall not say anything here is a different shape with its short sides together. Bothabout the less common bacteria which cause suppurative Streptococcus pneumonias and Neisseria meningitidismeningitis. Streptococcus pneumoniae and Neisseria are often seen in pairs. Streptococcus pneumoniae oftenmeningitidis are round bacteria or cocci, but forms short chains. The word streptococcus means aHaemophilus infuenzae is a rod-shaped bacterium or chain of cocci. Haemophilus influenzae is often of manybacillus. Streptococcus pneumoniae is Gram positive (see different shapes, some long and thin, some short and fatSection I 1.5), Haemophilus infuenzae and Neisseria (it is said to be pleomorphic, or many-shaped). In somemeningitidis are both Gram negative. We are therefore cases of suppurative meningitis you will see bacteria

‘.a,;, _^.,_ ,.:‘:,

Cerebral mil-aria 1 9.20blood has got into a specimen of CSF from a tom vesselin the brain or from the hole made by a lumbar punctureneedle.When red blood cells have been in the CSF for somedays they start to break down, and the yellow substancebilirubin is formed (see Section 8.8). This stains the CSFyellow and it is said to be xanthochromic. If you see thata specimen of CSF is xanthochromic, you should alwaysreport it. Besides old blood, a very high concentration ofprotein will also make the CSF slightly xanthochromit.9.20 Cerebral malariaIt is sometimes diEcult to tell if a patient has meningitisor cerebral malaria (see Section 7.34). In such patients athick blood film and a specimen of CSF are usually sentto the laboratory. The CSF is usually normal in cerebralmalaria, but the thick film will show malaria parasites.QUESTIONS1. What is CSF and’where does it come from?2. Why is lumbar puncture so important? Why is itnecessary to do lumbar puncture very carefully?3. How much protein is there in normal CSF? In whatdiseases is the CSF protein raised?4. Why is Pandy’s method so useful? How would youdo it?5. What are the common kinds of suppurative meningitis?How can you diagnose them?6. How would you look for: (a) Trypanosomes,(b) Mycobacterium tuberculosis in the CSF?7. What is a ‘bloody tap’?8. What is xanthochromic CSF?9. What changes would you expect to find in the CSFin a patient with virus meningitis?10. What are the signs and symptoms of meningitis in:(a) a young child, and (b) an older child or adult?

10.1 Why we examine the stoolsStools are some of the commonest specimens that cometo a laboratory. We usually look at stools to find parasites.These can be worms, or their ova, or protozoa (seeSection 1.14). Often the best way to tlnd out which kindof worm a patient has is to look for its ova in his stool.We do this by looking at the stool with a microscope. Wemix a little of the stool on a slide with a drop of salineand look at it under a coverslip. This method is called asaline stool smear.As with other specimens, the first thing to do with astool is to look at it carefully. You will see some thingsthat you will not see with a microscope. A stool from ahealthy person is brown and formed. By formed we meanthat the stool has some shape. An unformed stool has noshape. Abnormal stools may be yellow, green, black, oreven almost white. They may be soft (not formed) or mayeven be liquid. A useful way of describing stools is to usethese words:FormedSoftDiarrhoeicWateryThere is no blood or mucus on a normal stool. Mucusis the sticky white substance that comes from your nosewhen you have a cold. It is made by the epithelial cellscovering the inside of the nose. Mucus is also made bythe cells covering the inside of the gut. A little mucus ismade by a normal nose or gut. But, if much mucus ismade, it means that the nose or gut is diseased. Wheneveryou examine a stool, report whether youjind it normal ornot, and describe any abnormalities you may see.There are no pieces of undigested food in a normalstool, and there are no worms. Some of the worms thatyou may see in an abnormal stool have been drawn inFIGURE 10-l. Picture A shows Ascaris lumbricoides,or the roundworm. Ascaris is a large worm and may be25 cm long or more. It is smooth and white without anysegments, and, as its name suggests, it is round. You willalso see from Pictures E and F that both the hookwormand Trichuris are very small, thin, round, white worms.Trichuris is also called the whipworm. You will seethat one end of it is thin and the other end of it is quitethick.The tapeworm, or Taenia, may be a metre or more inlength, and is so long that only parts of it have beendrawn in Picture B. At one end it has a very small headwhich is fixed by hooks or suckers to the wall of the gut.The tapeworm is made of many segments (pieces) whichare all the same and which grow from behind the head.Close to the head the segments are very small, but asthey go down the worm they get larger. Mature segmentsare always breaking off from the end of the worm, eitherone at a time or several at a time, and are found in thestool. These mature segments have been drawn largerin Pictures C and D. You will see that the two speciesof tapeworm, Taenia saginata and Taenia solium, areslightly different. You can see these differences by takinga mature segment, pressing it between two slides, andholding it up to the light. You will see that most of thesegment is filled by the ovary, or egg sac, which has beendrawn black in these pictures. The ovary of T. saginatais shorter, straighter, and less branched than the ovary ofthe T. solium. The eggs of both these tapeworms lookexa.ctly the same. They are shown in Picture 4, FIGURE10-7.You may see any of the worms in FIGURE lo- 1 in thestool, and you are especially likely to see them when thepatient has been given some drug to kill them. Deadworms are unable to hold on to the gut and are easilypasseo in a stool. Other worms are sometimes seen, butthese are the important ones.First of all we shall describe the saline stool smear,which is the simplest way of finding parasites in the stool.Then come the ‘Cellophane’ thick stool smear, theformol-ether concentration method, and the ‘Sellotape’swab for Enterobius. When you write a report, say whichmethod you have used.Four ways of finding parasites in the gut10.2a One-The saline stool smearIt is easy to make a saline stool smear, but it takes a longtime to learn how to look at it well. Looking at a salinestool smear is difficult because there are so many differ-

The saline stool smear 1 10.2aent things to be seen in a normal stool. Many of themcome from the food that the patient ate. It is often difficultfor someone who is learning to be sure if somethinghe sees is a parasite, or if it is only a piece of the patient’sfood. We shall try to make this easier with many pictures.METHODTHE STANDARD SALINE FAECAL SMEAR. FIGURES 10-2 AND10-31. Wheil you ask an adult for a specimen of stool, givehim a ‘Polypot’ (ML 14~). a sheet of newspaper to put inthe latrine, and a stick or a strip of strong cardboard withwhich to put his specimen into the pot. Don’t givepatients universal containers for their stools. They getlost and they gre too expensive to lose. Use ‘Polypots’Ascaris lumbricoideswhich can be boiled or autoclaved and used again, asdescribed in Section 3.12.An easy way to get a stool specimen from a child is toput a smooth-ended glass tube into his rectum. Thisshould be 8 cm long and 7 mm in diameter and have itsedges smoothed in the flame as described in Section3.9. If a child has diarrhoea, the tube will soon fill withfaeces which can be looked at microscopically. Keep asupply of such tubes, and wash and boil them after use.Pieces of drinking straw or pieces of plastic tubing froma blood-giving set can also be used.If you have been asked to look for amoebae orGiardia. make sure that the stool you examine is so freshthat it is still warm. You will not find motile amoebae ina cold stool.Look carefully at the stool. Choose the mostabnormal-looking part of the stool to make your smear.Ascaris is a long, thin, unsegmentedthis is the headthe proglottids grow from behind /the head and are very small at firstabout 5 metres longEHookwormthis is what youF \ev.about 4 cm long two sk-les andboth these are small, thin roundworms : they are both nematodes : onlythe females are shown : the males areslightly differentFig. 10-l Some worms in the stool

T ,’10 1 StoolsIf there is mucus or blood on part of the stool, takesome of this part and make sure you report it.Put a drop of saline on a slide. It will be easier tomeasure out the right size of drop if you keep the salinein a ‘Polystop’ bottle.2. There are several ways of mixing the stool with thesaline. A wire loop can be used, so can an applicatorstick (orange stick), so can the stick that forms themiddle of a palm leaf. If the stool is hard and difficult tomix with saline, the best thing to use is a straightenedpaper clip with one end hammered flat. This makes alittle spatula, with which it is easy to break up thehardest stool. Make many of these ‘paper clip spatulae’,put used ones in a little jar of lysol, sterilize them, anduse them again.3. Mix the stool in the drop of saline. Stir (mix) untilthe smear is an even mixture of stool and saline. Thesmear must not be too thick or too thin. One way ofjudging the thickness of a smear is to put it on a newspaper.The smear should be so thick that you can onlyjust read the ordinary small writing of a newspaperthrough it. A smear of this thickness under a 22-mmsquare coverslip is called a standard faecel smear. Thesmear is too thick if you cannot read the newspaperthrough it. The smear is too thin if you can read thenewspaper through it very easily. Look at Figure 10-3.A standard faecal smear contains about 2 mg of stool.4. Put a coverslip on the smear. A polythene or ‘Cellophane’coverslip can be used as described in the nextmethod, but glass ones are better for thin films.5. Add a drop of Lugol’s iodine to one edge of thesmear. It will slowly run underneath. Iodine makes thenuclei of protozoa easier to see. Like saline, Lugol’siodine is best kept in a dropping bottle. You can leavethis out, and many people only use Lugol’s iodine whenthey specially want to look at protozoa. Other peoplealways make two smears whenever they look at a stool,one in a drop of saline :snd the other in a drop of Lugol’siodine.6. Seal the edge of the coverslip with a mixture of hotVaseline and paraffin wax. This is also shown in Figure7-14. Both the wire and the paraffin wax must be hot.BEGIN BY LOOKING ALL OVER THE SMEARWITH A VERY LOW POWER (x4) OBJECTIVE.Search it by the method shown in FIGURE 6- 14. You willeasily see ova with a very low power objective. Thensearch part of the smear with a low power (x 10) objective.With this objective protozoa1 cysts look like littleround bubbles or balls of glass. You will know that theymake a smear fromthe most abnormalpart of the stool straightened paper clip,applicator stick, orwire loopa Polystopbottlestir the stool in thesaline until you canend tlattened--r/slide 1 if you are looking for nprotozoa, put a dropLugol’s iodine at oneedge of the slide : itwill run under thein a Polystop bottleFig. IO-2 Making a saline stool smearseal the edges of thecoverslip with hotVaseline and paraffinwax mixture

,L- ;.:

4. Leave the smear for about half an hour in a warmdry place. A dry incubator is best. Some of the water inthe smear will evaporate, and the stool will clear in theglycerol.5. Search the whole film with a low power x4 objectiveand a x 10 eyepiece. Count the number of each kindpresent.glycerol and malachitegreen solutiun, seeSection 3.221this is a THICKfilm, so useenough stoola loop or applicator4bpenci!0stickDress the stool towards theedge of the cellophane usingthe flat end of the rubber on apencil : a rubber bung orcork can also be usedThis is a method for ova, and it is not good for protozoa,because the glycerol kills them and stops themmoving. If you want to find protozoa, make a standardfaecal smear in saline.There is no point in sealing these ‘Cellophane’ coverslipswith Vaseline and paraftin wax, because they aresoft, and it will not hold them. They can, however, beused with an oil immersion objective. But because theycannot be held by sealing with Vaseline, objects moveduring focusing. ‘Cellophane’ coverslips can also be usedinstead of glass ones for other methods. If used dry, theycurl up, so soak them in saline first.The important part of this method is to have the rightkind of film. It must be ‘Cellophane’ of the right thickness.Thin, unscratched polythene film from an ordinarypolythene bag can be used, but it is not nearly so good.Because it is not wettable (water runs off it), you willhave to mix the glycerol with the stool on the slide beforeputting on a polythene coverslip. If you run out of glasscoverslips, try using polythene ones., ,, I-.--- .- ,I_--_-.-_Iput the slide on something soft while you are pressing, so that it doesnot break : a pile of newspaper s uares is a good thing to rest it ona slide isabout~~“~!~~~~~~~~e4 LEAVE THE SLIDE FOR HALF AN HOURthan a slide search the WHOLE film at a low magnification( x 4 objective and x 10 eyepiece)Fig. 1 O-4 The ‘Cellophane’ thick stool smear METHOD4 mm x 4 mm x 4 mm. The important thing is that muchmore stool is needed than for a standard saline smear.2. Using a pair of forceps take one of the soaked‘Cellophane’ coverslips out of the stock bottle (seeSection 3.22a). and put it down on top of the stool.3. Press the ‘Cellophane’ down firmly on the stoolwith the rubber on the end of a pencil. A rubber bung orcork can also be used. Carefully spread out the stoolunder the ‘Cellophane’. It should reach nearly to thesides of the ‘Cellophane’, but need not get to the end.The best thickness for the smear is important, and youwill have to learn it with practice. Glycerol clears quite athick smear./10.3 Three-The formal-ether concentration testIf there are only a very few parasites in a stool it takes avery long time to find them even in a thick stool smear.Parasites can be found more easily by doing a concentrationtest. This takes away most of the normal parts of astool and leaves the parasites. We shall describe the‘formol+ether concentration test’. Ether is expensive andoften difficult to get. Fortunately petrol can be usedinstead and seems to work equally well. This methodconcentrates both the eggs of worms and the cysts ofprotozoa. It does not, however, concentrate protozoa1trophozoites.THE FORMOL-ETHER (OR PETROL) CONCENTRATION TEST,FIGURE 10-51. Add a few ml of water or saline to the container(bottle or box) in which the stool was sent to the laboratory.2. Stir the stool and the water together with an applicatoror clean glass rod (see Section 3.91. By stirring thestool in this way you will get a thin suspension of stooland saline.3. Put two layers of surgical gauze (pieces of gauzeone on top of the other) in a small plastic funnel. Surgicalgauze is the thin white cotton cloth that is put onwounds. Pour the stool suspens.an through this into acentrifuge tube. The large pieces will be left behind on

spin the suspensionfor about five minutes.;‘:.thin stool suspension.,51 ,,.: +:‘f’:“:“‘,.. ‘c: .,:t..r -‘-..’..*,,,a ::- :..wav ,..,:.filteredBe oq i :.-suspension:,..:,tfGsuparna>nt, ,’,.. ._,,. :- ~ ?’.;,;$;! /tezzgzJgpT::’i.’ , 7 ~of form01 / .’ \saline c- - :J\hs\ / __:.. \ +----this is ether, but\ pour away thecGar~uperGrata_nt ,.--.. _ - -.~ shake hardAlp.

10 1 Stoolsthe gauze, and a thin stool suspension will go throughinto the centrifuge tube.4. Spin the centrifuge tube for about 5 minutes. Anel$ctric centrifuge is shown in the drawing, but a handcentrifuge will do.5. You will be left with a brown deposit and a brownishcloudy supernatant fluid. If you have used the rightamount of stool there will be about f ml of deposit aftercentrifuging.6. Pour away the supernatant fluid into lysol.7. Fill up the centrifuge tube with saline or water.8. Mix up the contents, either by stirring with theapplicator stick or by shaking with a cork in the top of thetube.9. Centrifugeagain.10. If the supernatant is not clear, go back to step 6,pour away the supernatant, add more water or saline, andcentrifuge once more. If, after this, it is still not clear, goback to step 6 a third time. If the supernatant is then clear,goontostepll.11. Pour away the clear supernatant.12. Add 10 ml of formol saline.13. Wait 5 minutes.14. Add 3 ml of ether (or petrol).15. Shake hard.16. Centrifuge for 2 minutes. This should be at 1,500revolutions (turns) a minute. You will not be able tomeasure this, but it is about as fast as a hand centrifuge or asimple electric centrifuge will go.17. You will see four layers in the tube: a top, clear,yellowish, ether or petrol layer; a narrow, middle, thickbrown layer; below this there is a thin, brown layer; at thebottom is a little layer of deposit.18. Put an applicator stick [or a loop) into the tube andmove it round the inside wall-this stops themiddle, thick brown layer from sticking to the side of thetube.39. Tip away the supernatant, except for the deposit atthe very bottom of the tube.20. Add a drop of Lugol’s iodine to the deposit.21. Using a Pasteur pipette put the deposit on a slideand look at it under a coverslip. You will see many more ovaand cysts than there are in an ordinary saline smear of thesame specimen, but you will not see any protozoa1 trophozoites.Search the whole ofthespecimen as in Figure 6-l 4.‘10.4 Four-The ‘Sellotape’ swab for Enterobiusvermicularis (FIGURE 10-6)Enterobius vermicularis is also called the threadworm. Itis a small, thin, white worm which lives in the largeintestine. At night the female threadworm crawls (moves)out of the anus (the opening through which the stools arepassed) and lays her eggs on the skin in the fold betweenthe buttocks (the parts of our bodies that we sit on). Theeggs of this worm are thus more easily found on the skinround the anus than they are in the stool. The easiest wayto find them is to use a ‘Sellotape’ swab. ‘Sellotape’ is thetransparent (clear) sticky tape (a tape is a long, thin, flatpiece of paper or cloth) that is used to stick parcels andletters. If a piece of this transparent sticky tape is pressedon the skin round the anus the eggs of Enterobius willstick to it. This tape is then stuck to a slide and lookedat with a microscope. The tape works like a coverslip,and the eggs of Enterobius can be seen through itclearly. They are shown in Picture 6 in FIGURES 10-7and 10-8.METHODTHE ‘SELLOTAPE’ SWAB FOR ENTEROBIUS VERMICULARIS.FIGURE 10-61. Take a strip of ‘Sellotape’ and fold it round thebottom of a test tube or tongue depressor with its stickyside outwards.2. Press the sticky surface of the ‘Sellotape’ firmlyon the skin round the anus. Flatten the folds of skin asyou press. The eggs on the skin will stick to the‘Sellotape’.3. Cut off the middle part of the strip of ‘Sellotape’which has been pressed on the skin.4. Put three or four drops of xylol on the slide andstick the middle part of the tape sticky,side down on topof it. Look at the strip with a low power objective, andyou will easily see the ova of Enterobius vermicularis.Enterobius vermicularis is not a very harmful worm. Itis usually found in children and makes a child’s anusitch, so that he wants to scratch it, especially at night.MOSTLY ABOUT HELMINTHS10.5 Some common ova (FIGURES 10-7 and 10-B)In FIGURE 10-7 the common parasites are drawn asdiagrams. These diagrams show what you might or couldsee, and the parts of the figure are not always drawn toscale (the right size compared to one another). In FIGURE10-8 these same ova are shown as drawings. They havebeen drawn as nearly as possible to the same scale, andwere made using the Olympus Model K microscope; soyou should be able to see everything in these drawingswith your own microscope. FIGURES 10-7 and 10-8 arenumbered in the same way.This section describes the parasites that are commonin Africa. In the place where you work other parasitesmay be common, and you will have to ask other laboratoryivorkers which these are likely to be.HookwormThere are two species of hookworm called Ancylostomaduodenale and Necator americanus. Although the adultworms look different, their ova look the same. They havethus been labelled ‘hookworm’. The adult hookwormlives in the small intestine where it is hooked on to themucosa by its mouth. As you read in Section 7.6, the

testtube-I piece ofSellotapelay the middle piece of 1the tape, sticky side downon a slideHIGH POWERt/ei&cpress the tape roundthe patient’s anus7cut off the middle piece/of the tape where theova have stuck, slidethe Sellotape workslike a coverslipSome common ova 10.5young worm) is formed (Picture lh). This then bursts outof its shell (Picture lj) and becomes the free larva. This isthe first stage larva. An embryo is said to become alarvawhen it bursts out of its shell.Most of the hookworm ova that you will see will be inthe early morula stage (Pictures lb, c, d). This is becausethere is not usually enough time before you look at thestool for the hookworm ova to grow to the stages shownin Pictures lg, h, and i. But, if the stool has been waitingin a warm place before you look at it, you may find thestages shown in Pictures lh and i. The hookworm larva(Picture li) is very like the Strongyloides larva (Picture7). For this reason both the hookworm larva (Pictures 8and li) and the Strongyloides larva (Picture 7) have beendrawn together for comparison at the bottom of bothFIGURES 10-7 and 10-6. We shall say more about thedifference between the hookworm and Strongyloideslarvae when we come to describe Strongyloides.A few hookworms do not harm a patient, but manyhookworms cause so much bleeding that they may givehim a severe hypochromic anaemia. It is thus importantto find out, not only that the patient has hookworms, butalso how many he has. This can be done quite easily bymaking a standard thin faecal smear as shown in FIGURE10-3 and then counting all the hookworm ova seen in thefilm. This means searching the whole area of the coverslip,as shown in FIGURE 6- 14. It also means that the filmmust be standard-that is it should only just be possibleto read newsprint through it. If a child only has 20 ova ina standard film, his infection is mild and it may not benecessary to treat him. Above 20, and certainly above40, he should certainly be treated. When there are morethan 70 ova in the standard film, there are so manyhookworms in the gut that they cause enough bleeding tomake the occult blood test positive (+) (see Section10.10). When it is strongly positive (++) there will neverbe less than this number of ova in the stool, if hookwormsare the only cause of the bleeding. If therefore theoccult blood test is strongly positive (++) and yet thereare less than 70 ova in a standard film, some other causefor the bleeding must be looked for.if you have difficulty seeing the ova because there is so muchdebris, let a drop of xylol run under the SellotapeFig. 10-6 The ‘Sellotape’ swab for Enterubiu vermicularismucosa bleeds a little where the hookworm bites it. Thefemale hookworm lays many ova. Just after they are laidthese ova look like Picture la and have one large roundcell in a thin, clear shell. The cell soon divides into two(Picture lb), and then into four (Picture lc). After a fewmore divisions it becomes a ball of small cells (a morula)as in Picture Id. The ball becomes empty, as in Picturele, and a depression (hole) forms in one side. A rod ofcells grows into the middle of the ball, and, after furthergrowth (Picture If and lg), the completed embryo (veryAscarisiumbricoidesAs you will see from FIGURE 10-7, the ova of Ascaris areabout the same size as those of the hookworm. But,instead of having a thin, smooth, clear shell like hookworm,Ascaris ova have a thick, rough, dark shell, Picture2a shows what the ovum looks like from on topthick,dark, and rough. If you focus on the middle of theovum you can see it as if it were cut in half as in Picture2b. This is a fertile ovum-that is, one which is going togrow into an Ascaris worm. It has a thick shell, and thereare few granules in the ovum itself. Some ova are notfertile-that is, they will not grow into an adult worm.These infertile ova are longer and narrower than thefertile ova and have a thin shell and many round brightgranules (little round balls) inside them. These are shownin Picture 2c. There is one more kind of Ascaris ovm-

these are the stages YOUshellthe decorticate ovum ofAscaris is sometimesempty hookworm shellmistaken for theovum of the hook- 3 Trichurisworm : Ascaris hasa thick shell, andthe hookworm avery thin oneclearPlate 73 0thickthis ovum is saidshellto look likea tea trayECORTICATE‘fewgranules 5 - Schistosotia mansoni 6 EnterobiusPlate 85Py+-shortmouthfold or notch in it like thusthese larvae can be distinguishedmore easily when they have beenthis is the hookworm larva in Picture 1 i above drawn much larger so that it can be compared with the larva of StrongyloidesFig. 10-7 Some common ova-a diagramthe decorticated ovum. Decorticated means that theovum has lost its skin or cortex. It is like an orangewithout its skin and is shown in Picture 2d. All these ovaare reported together as ‘Ascaris ova’; there is no need todistinguish one kind from another.With the hookworm we saw that the ovum started as asingle cell (Picture la) and grew through many stages tobecome a larva (Picture li). We said that all these stagesmay be found in the stool, but that the later ones are notvery common. Ascaris grows through the same stages,but it grows so slowly that only the single-cell stage isfound in fresh stools.

;: “,Some common ova1 10.5TrichuristrichiuraThis is a very easy ovum to recognize--it is longer andnarrower than the other ova and has clear lumps at eachend. It is sometimes said to look like a tray, the lumpsbeing the handles. Like the Ascaris, it is only seen in theone-celled stage. Unlike both hookworm and Ascaris,there is only one kind of Trichuris ovum. This is shownin Picture 3a.TaeniaThere are two common kinds of tapeworm: Taeniasolium and Taenia saginata. Like the two kinds of hookworm,both their eggs look the same. They are nearlyround with a smooth, dark, thick shell. Inside the ovumare six little hooks. When the worm grows these will bethe hooks of the head with which it fixes itself to the wallof the intestine. These hooks have been drawn in Pic-.ture 4a. You can usually see the hooks if you lookcarefully with a high power objective.SchistosomamansoniThis is the largest egg you will find in tht: stool. As yousee from Picture 5, the ova of S. mansoni differ a little inshape, but they all have a sharp spine (a thorn or spike)sticking out of one side. The egg of S. mansoni is verylike the egg of S. haematobium (Picture 1, FIGURE 8- 10)except for the place of its spine. S. mansoni has its spineat one side (a lateral spine); S. haemarobium has its spineat one end (a terminal spine). S. nunsoni usually lives inthe gut, and its ova are found in the stool. S.haematobium usually lives in the bladder, and its ova arefound in the urine. Therefore you will usually find lateralspined ova (S. mansonz) in the stool and terminal spinedova (S. haematobium) in the urine. But you will occasionallyfind S. mansoni in the urine and S. haematobiumin the stool.In Picture 5, FIGURE 10-8, you will find that the ovaof S. mansoni have been drawn just as they are seen inthe stool, and it is difficult to find the various parts of theembryo schistosome. But, in Picture 5, FIGURE 10-7, theparts of the embryo are shown as a diagram, and you cansee them more clearly.EnierobiusvermicularisThis worm is also called the threadworm, and its ovaare not commonly seen in the stools. They are betterseen by the ‘Sellotape swab method’ which is descibedin Section 10.4. The ovum of Enterobiiis isthin and clear like that of the hookworm, but, unlikethe hookworm, it is flat on one side. As with thehookworm, many stages of development are seen,but only three are drawn here. The single cell ovumhas been drawn in Picture 6a, the morula (ball ofcells) stage in Picture 6b, and the larva about to hatchin Picture 6c.Strong yloidesstercoralisThis worm lives in the small intestine, and the larvaehatch very soon after the female has laid her ova. By thetime the stools are passed they have had time to hatch.This means that Strongyloides are not seen in the stool,but Strongyloides larvae are seen. If actively movinglarval worms like those drawn in Pictures 7 and 8 areseen in the stool, they are either hookworms orStrongyloides. They will only be hookworm if the stoolhas stood about on the bench and the hookworm eggshave had time to hatch. Larvae in the stools are thususually Strongyloides. It is quite easy to tell hookwormfrom Strongyloides by looking at their mouths.Strongyloides has a short mouth, and the hookworm hasa long mouth. To see the mouths more clearly, use thehigh power objective and not too much light. Strongyloidesalso has a notch in its tail.Some other thingsSome of the other things that may be seen in a stool havebeen drawn in Picture 11, FIGURE 10-8. The little dotsand rods that are seen all over this picture are the normalharmless bacteria that are seen in all stools. Pictures 1 la,b, c, d, e, f, are all parts of plants, such as cabbage orbanana that the patient has eaten as his food. There arealso many other parts of plants with quite differentshapes which there is no space to draw here. Some piecesof plants, such as those drawn in Pictures 1 la, c, and fare so big that only a part of them has been drawn. Spiral(spring-like) cells, such as those in Picture 1 Id are oftenseen. Picture 1 lg is an air bubble; this is a small onetheycan be very big.Pictures 1 lh and 1 li are both fibres (little threads) ofmeat that the patient has eaten but which have not beencompletely digested. A few meat fibres are often seen.But, if many meat fibres are seen, this is abnormalbecause it means that the patient is not digesting his meatas he should. If you see many meat fibres, report ‘manymeat fibres seen’. Meat Bbres are easy to recognize. Theyare brownish-red like meat, and they have rounded cornerswhere they have been partly digested. Meat fibresalso have ‘cross striations’: that is, they have many smalllines going across the fibre. Look for cross striationswith the high power objective,Little drops of oil or fat are often seen in the stool;these are shown in Picture 1 lj. They are very ‘bright’(refractile), completely round and of varying sizes. Oildroplets arc often -muddled up with cysts. Some cystshave been drawn in Picture 1 lk. You will see that cystsare not quite so ‘bright’, and they are not quite so round.This is an unstained saline smear; so you cannot see thenuclei easily. If they were stained with iodine they wouldlook iike Picture 9. An amoebic trophozoite has beendrawn as Picture 1 II. Red cells, white cells or pus cells,bacteria, yeasts, and Blasrocystis are also shown.Something more must be said about Blastocyst!~hominis which is a common and harmless yeast. The

1 hookworm Plates 65 - 69 2 Ascaris Plates 71 - 75 3 Trichurislate 70Schistosoma 5mansoni4 Taenia f’latS 79 8180Plate 85Vteral /or side spineeverything here is drawnapproximately to scale0notch inpointedtail-.9 pmtozoathese have been stained with iodine toshow the nuclei etc.10 other cellsmouthlongmouthPlate 8611 some other things to be seen in the stoolclear centralor vacuolespaceFig. 10-8 Some common ova-a drawing

important thing about these organisms is that they caneasily be mistaken for protozoan cysts. They are more orless round and of varying size. The smallest are about thesize of a red cell and the largest several times as big.Most of the organism is tilled with a large, clear, emptylooking,spherical (ball-like) space or vacuole. This largevacuole pushes the nucleus and the rest of the cytoplasmto the edge of the cell. I?lasroqrs~& has been drawn inPictures 10 and 11 in FIGURE 10-8. Learn to recognizeBlas&q&s, and you will not muddle it up with protozoancysts.10.6 Some more ova (FIGURE 10-g)Most of the parasites and their ova we have described sofar 7 are common in Africa, and many of them are alsocommon in other parts of the world. Some of the ovadescribed in this section are rarely, if ever, seen inAfrim although they may be very common in othercountries, especially countries in the Far East. Youshould ask which ova you are likely to see and shouldnot waste time learning to vecognize ova that are notfound in the place where you work.The first four are the ova offlukes. A fluke is a kind oftrematode worm (see Section 1.14) which is short, flat,broad, and thin, and is shaped rather like a leaf.Picture A, FascioIopsis buski. This is a large flukewhich lives in the liver and is common in Eastern Asiaand in the South-West Pacific. The egg is very largeand has an operculum at one end. An operculum is adoor or weak part of a shell through which the embryocan escape when it is mature and ready to leave the egg.Picture B, Heterophyes heterophyes and Metagonimusyokogawai. Both these flukes live in the intestine, and, astheir eggs are almost exactly the same, they are describedtogether. H. heterophyes is found at the mouth of theNile, in Turkey, and in the Far East. M. yokogawai iscommon in the Far East. These ova are small, lightbrown, and have an operculum.Picture C, CIonorchis sinensis. This fluke lives in thebile passages and is found in the Far East, especially inthe region of the China Sea. The ova are small and have athick, light brown shell with an easily seen operculum.The ova of this species may be hard to distinguish fromthose of H. heterophyes and M. yokogawai.Picture D, Paragonimus westermani. This Auke isfound in the lung, and its ova are seen in the sputum. It isfound in the Far East and Western Pacific and also inWest Africa. The ova are large and brown with a thickwall and ffattened operculum.Picture E, Schistosoma japonicum. This trematodelives in the intestine and is found in parts of the FarEast. Its ovum is smaller than that of S. mansoni and hasa small curved spine at one side. The spine is in a slightdepression (hole or cup) and may not be easy to see.Picture F, Hymenolepis nana. This is the dwarf C; :rysmall) tapeworm and is common in many parts of theworld. Like Taeniu its ova have a thick shell, but its wallsappear double. Between the inner and outer walls aresome thin twisted threads. Inside are three pairs of hooks.1 /operculumBG/ operculum /operculum+----Plate 84 -small lumpHETEROPHYESHETEROPHYESAZ?9C :LONORCHISSINENSISe ovum of S.japonicumoften surrounded bypieces of tissue like this PARAGONIMUS WESTERMANIFASCIOLOPSIS BUSKI Plate 78SCHISTOSOMA JAPONICUM ‘ , I 4 40 20 40 60 80 100operculumHYMENOLEPIS NANA DIPHYLLOBOTHRIUM LATUMFig. 10-S Some less common ovawhich is sometimes eaten by manHETEPODERARADICICOLA

;,_=,“,I’ ,. -.‘1 _,-,10 1 stoolsPicture G, Diphyllobothrium la&m. This is the fishtapeworm which is found in parts of Russia, Central andSouthern Europe, the Middle and Far East, LatinAmerica, and Australia. It is quite a large, yellow-brownovum with thick walls and an operculum.Picture H, Heterodera radicicola. This nematode is aparasite of plants and is only found in the stools when apatient has eaten plants which are infected with it. It isquite harmless.rPROTOZOA10.7 E. histolytca and E. co/i (FIGURE lo- 10)Protozoa are very much smaller than the helminths, sothey are described separately and drawn larger on adifferent scale.There are many protozoa in the gut, but the two whichcause disease are Entamoeba histolytica and a flagellatecalled Giurdia lamblia which is described in Section10.10. The harmless amoebae are Entarnoeba coli,Dientamoeba fragilis, Endolimax nana, and Iodamoebabutschlii. We shall describe Entamoeba coli in somedetail because this is the organism which is often confusedwith Enramoeba histolytica. Earlier on you readabout Escherichia coli, which is often shortened to Esch.coli. This is a bacterium, not a protozoon, and is thusquite a different organism. ‘Coli’ means belonging to thelarge gut or colon, which is where both these organismsare most often found.Entamoeba histolytica may invade (harm and gointo) the wall of the colon and cause a kind of bloodydiarrhoea called amoebic dysentery. The stools of suchpatients will contain actively moving trophozoites withred cells inside them. In many people, however, E.histolytica seems to cause no disease and ‘to live harmlesslyin the upper part of the colon. The stools of thesepatients look normal and contain only cysts. There is,however, every intermediate stage between a very sickpatient with severe diarrhoea caused by many trophozoitesand a person who passes a few cysts and seemsperfectly well. Thus a patient might be only mildly ill andpass soft stools containing a few trophozoites of E.histolytica.FIGURE 10-8 shows you what the trophozoites andcysts of E. histolytica look like in a saline smear of thestool (Picture 11 in this figure). In FIGURE lo- 10 theseorganisms have been drawn very large as diagrams-seehow large a red cell is by comparison (Picture f). Thetrophozoites of E. histolytica and E. coli (Pictures a andg) are at the top of the figure. You will see that thecytoplasm of the trophozoite of E. histo!vtica has a clearouter part or ectoplasm and an inner endoplasm whichcontains small granules. In this it is unlike the trophozoiteof E. coli, the cytoplasm of which is the same allover and is much less clear and more full of granules.Many of these granules are the remains of bacteria whichthe amoeba has eaten and which are more obvious in E.coli than in E. histolytica.In both species the nuclear chromatin is spread roundthe edge of the nuclear membrane and is also gatheredinto one or more small lumps or karyosomes. Entamoebuhistolyticu has finer chromatin round the edge ofthe nucleus, and its karyosome is usually in the middle.Entamoeba coli has coarser chromatin round the edge,and its karyosome is usually towards one side. TheEntumoeba kind of nucleus is quite different from thenuclei of other species of amoeba, which have their nuclearchromatin arranged in djfferent ways, as shown atthe bottom of FIGURE lo- 10. ti3,ou want to see the detailof the nuclear chromatin, mak&,an iodine film, seal itwith Vaseline and paraffin wax, :~$d use an oil immersionobjective. +*Pictures b and h in FIG&E lo- 10 show the amoebaeturning into cysts. It is not easy to tell one species fromthe other at this stage. The lower three pictures (Picturesc, d, e and i, j, k) show the cysts. The mature cysts shownin Pictures e and k are commonly seen. Young cysts areless common. The cysts of E. histolytica seldom havemore than four nuclei; the cysts of E. coli usually havemore than four nuclei and may have eight. The youngcysts of both species sometimes have bars or rods insidethem. These are called chromidial bars. The chromidialbars of E. histolytica are large, rounded, and easily seen;the chromidial bars of E. coli are smaller and pointed. Itmay sometimes be difficult to tell the cysts of E. colifrom those of E. histolytica. The number of nuclei andthe shape of the chromidial bars (if there are any) are thethings to look for-they can easily be seen when the filmis stained with iodine.There are two important ways of telling the trophozoitesof E. histolytica from the trophozoites of E. coli.Only E. histolyticu ingests (eats) red cells, and only E.histolytica moves in the way shown in FIGURE lo- 11. InPicture A in this figure an E. histo(vticu trophozoite hasbeen drawn as if it was about to cross a line X-Y on theslide. Inside it are two red cells. In Picture B it has putout a clear pseudopodium (a foot) as shown by thearrow. In Picture C the inner granular part of the cytoplasm(the endoplasm) has moved into this foot and withit one of the red cells. In Picture D the amoeba hasmoved in its ‘tail’ as shown by the black arrow, and at thesame time it has put out a new pseudopodium, as shownby the white arrow. In Picture E the amoeba has movedin its ‘tail’ once more. In this way E. histolytica changesits place-it will soon have crossed the line X-Y on theslide. The way E. histolytica moves has been called the‘progressive directional crawl’. A crawl is a slow way ofmoving. It is a good name because E. histolytica movesas if it wanted to get somewhere. E. coli does not move inthis way-it slowly puts out small pseudopodia, whichare not clear like those of E. histolytica. It does notchange its place, and it does not ingest red cells. ‘Progressivedirectional crawl’ is a very important way ofdistinguishing E. histolytica, but you will only see it in afresh warm stool very soon after the patient has passed it.This is why warm stools a@ so important in looking forthis organism.._

E. histolytica and E. co/i 1 10.7E. histolytica1 ectoplasmE.coli,vacuole(empty space)TROPHOZOITESendoplasm:ellsnucleus-nucleus with centralkaryosome andparticles of chromatinhfood particlesbacteria ingestedby the amoebaeAMOEBAE TURNINGINTO CYSTS(pfecystic forms)large found endedchromidial barsYOUNGCYSTSthin sharpchromidialbarsPlates 88 & 89four nucleiMATURECYSTSeight nucleik&Z?@these nuclei will onlyjust be visible in saline,and are much more easilyseen in an iodine filmA RED CELL DRAWN TOTHE SAME SCALEthese are drawings of the amoebaewhen stained with a special blackstain called iron haematoxyliniron haematoxylinsaline or iodineredrawnafter BeldingDIFFERENTSPECIES OF AMOEBAE HAVE DIFFERENT NUCLEIto see these detailqmake an iodinethese are diagrams and Don’t worry about identifying these species, thepreparation, seal it with Vaseline andare not to the same scale important thing about them is that they are notwax, and use an oi! immersion objectiv~~~~mo~@Entamoeba histolytica Entamoeba coli Endolimrx nana lodamoeba butschlii Dientamoeba fragilisFig. lo-10 Entamoeba histofytica and Entamoeba co/i

THIS IS THE PROGRESSIVE DIRECTIONAL CRAWL Plates 86 & 87the amoeba is just it has put out a the endoplasm has the amoeba.is pulling the erlrdoplasm has flowedabout to move clear pseudopodium flowed into the in its tail and is putting into the ectoplasm onceof ectoplasm pseudopodium out another pseudopodium more and it is pulling inits tail againThis is a saline preparation from a WARM stool : it has not been stained with iodine, so the details of the nuclearchromatin are not easily seenFig. 10-l 1 The ‘progressive directional crawl’ of E. histolyticaIf you are wondering what something is and it has redcells inside it, it might be E. hisfoZyf&, and it might be amacrophage. It could not be E. coli. A macrophage isusually a monocyte that has come from the blood. Macrophagemeans ‘big eater’, and macrophages may eat redcells. But macrophages do not put out pseudopodia.They do not move like E. histolyticu, and they do nothave nuclei with chromatin round the edge like theentamoebae.10.8 Bacillary and amoebic exudates (FIGUREIO- 12)Very bad diarrhoea with blood in the stools is calleddysentery. There are several kinds of dysentery, but the twomost important kinds are bacillary dysentery and amoebicdysentery. Bacillary dysentery is caused by bacteria ofthe genus Shigella. As we have seen, amoebic dysenteryA baciliary B amoebicis caused by E. histolytica. The stools of these two kindsof dysentery are different and show different exudates.A stool from a patient with bacillary dysentery isusually watery and is stained with blood. If the dysenteryis very bad the stool will have in it pieces of the mucosaof the gut. The mucosa of the gut is its inside surface.When a saline smear is made and looked at with a microscope,it will look like Picture A, FIGURE lo- 12. Therewill be many red cells, many pus cells (polymorphs), andprobably some macrophages. Some of these may havered cells inside them, but, unlike E. histolytica, they willnot be motile (moving).A stool from a patient with amoebic dysentery willprobably be soft. You may see blood in it, and you willalmost always find red cells when you look at a salinesmear with a microscope. A saline smear of an amoebicexudate has been drawn in Picture B, FIGURE lo- 12.You will see the trophozoites of E. histolytica with redrE.histolyticaCharcot -some of these cells have 1come from the mucosa ofthe gut\pus cells crawl ’ ingested red cellsFig. 1 O-l 2 Bacillary and amoebic exudates

.-.i- ; .>_; ,’ .:Identifying E. histolytica 1 10.9cells inside them. There may be a few pus cells, but therewill not be nearly so many as in a bacillary exudate. Ifyou watch carefully you will see the ‘progressive directionalcrawl’ described in Section 10.7. Sometimes youwill see small sharp-pointed crystals called Charcot-Leyden crystals. These are more often seen in amoebicdysentery than in any other disease. This is a typicalamoebic dysentery exudate, but in many patients withamoebic dysentery all you will see are a few amoebaeand some red cells.10.9 tdentifying E. histdyticaThis means 6ndmg and being able to say with certaintythat an orga;rism is E. histolytica. As we have seen, thismay not be easy, so here are some rules to help you.Always look at a fresh warm stool as soon as it ispassed, and make smears from several parts of it, especiallythose with blood or mucus on them. If the diagnosisis is in doubt, look at several stools, if necessary, afterthe patient has been given a purge. Don’t report on oneparasite only, but look at several before making up yourmind what they are. Make a smear in iodine so that youcan identify the Entamoeba kind of nucleus more easily.Try to find the size of any trophozoites or cysts you see.This is best done with a special eyepiece micrometer(size measurer), but you will have to do as best you canby comparing the organisms you see with red cells.By using these methods you may report E. histolyticaas being present if you find any of the following:1. Trophozoites showing a ‘progressive directionalcrawl’ and also containing red cells inside them.2. Trophozoites bigger than 12/1m with a fairly clearcytoplasm and a progressive directional crawl or theEntamoeba kind of nucleus (or both).3. Cysts bigger than 10 /cm containing not morethan four of the Entamoeba kind of nucleus. They areeven more likely to be E. histolytica if they containrod-shaped chromidial bars.When you report on a specimen which you haveexamined for E. histobtica, describe what the stool lookslike, give the genus and species of the parasite found, saywhether you found trophozoites or cysts, record whetherthe trophozoites contained red cells or showed the‘progressive directional crawl’, or whether the cysts containedchromidial bars. Say what kind of exudate youfound. The person who is looking after the patient willthen be able to tell if the amoebae are causing disease oronly living harmlessly inside the colon. As always, if youare not sure what you have found, say so.10.10 Giardia lamblia and Trichomonas hominisAmoebae move by putting out pseudopodia, but otherkinds of protozoa can move in other ways. Some protozoahave a few long hairs or flagella which they moveabout very fast like a whip. The word flagellum meanswhip. Protozoa which move with flagella are calledflagellates. The trypanosome described in Section 7.36TROPHOZOITETROPHOZOITEfrom underneath from the sideflagella \CYSTsucker or shieldabasal bodyPlates 90 - 93drawn from films stained withto show the detailed structureFig. lo- 13 Giardia lamblia,‘\here is a redcell drawn tothe same scaleiron haematoxylinis a flagellate: so is Trichomonas vagirzalis described inSection 11.8. Several flagellates are found in the stools.The most common and the most important stool flagellateis Giardia lamblia, which is shown in FIGURE lO- 13.Even though it is only one cell, this flagellate has severaldifferent parts and is a very special shape. You will seethat it has several flagella and a Rat area or sucker at oneend. Inside it are two curved rods and also two nuclei.Giardia lamblia forms oval (egg-shaped) cysts with athin wali. Inside the cyst there are four nuclei and somecurved bars which can easily be seen in an iodine film.Giardia lamblia is the only intestinal (gut) flagellatewhich causes disease in man. It lives in the small intestineand can cause diarrhoea. This diarrhoea is usually of aspecial kind called a fatty diarrhoea or steatorrhoea. Thestools of a patient with steatorrhoea are said to be ‘pale,bulky (there is a lot of them), and offensive’ (they smellespecially bad). They are also often frothy (they containbubbles of gas). The stools are like this because largenumbers of Giardia in the small intestine stop food andespecially fat from being absorbed properly. It is thisunabsorbed food in the stools which makes them ‘pale,bulky, and offensive’. Other diseases can also cause thiskind of stool, but giardiasis (the disease caused byGiardia) is an important disease to recognize because itcan so easily be treated with mepacrine tablets.Two other flagellates may be seen in the stools. Theyare Chilomastix mesnilii and Trichomonas hominis,neither of which are believed to cause disease. Both moveA10

‘10 1 Stoolsabout very actively and may be mistaken for G. lamblia.One way to tell them apart is by the way they move.G. lamblia moves in a rapid, jerky progressive way andmoves from one part of the field to another. It is sometimessaid to move by turning over and over like a fallingleaf. C. mesnilii moves in rather a similar way, whereasthe movement of Trichomonas hominis is irregular inthat it does not move across the field of view. Chilomastixmesnilii forms cysts, but Trichomonas hominis doesnot. If therefore you see a flagellate and are not sure whatit is, look for cysts. The cysts of G. lamblia are veryspecial, and once you have seen them you will notmistake them for anything else. Look for the oval shape,the thin cyst wall, and the curved bars. You will need ahigh power or an oil immersion objective, and you mustcarefully adjust the condenser to give the best light. Theadd 200 mg of thepowdered mixture oforthotolidine andbarium peroxideX,-i -------.cysts will be easier to examine if you seal the coverslipwith Vaseline and paraffin wax mixture while you areusing the high power, or the oil immersion objectives.It is easy to say if a micro-organism is a flagellate,because flagellates are the only common small protozoathat move around fast in the stools. If you know thatsomething is a flagellate but are not sure if it is G.lamblia, T. hominis, or another flagellate, report ‘Flagellatespresent’. If the patient has diarrhoea ?,rld you report‘Flagellates present’, the patient should be given mepacrinetablets, He probably has giardiasis.10.11 Occult bloodWhen a patient bleeds from his skin or his nose, theblood is easily seen. But when a patient bleeds a little intothis is the cork of thebottle into which thepiece of tin is stickingthis piece of tin is her 1tdown the middle and pointedat both ends->this is the mixtureorthotolidine andbarium peroxideofcan be used, socan astraightenedout paper clipshout 5 mlthis is the patient’smake up fresh solutionNEGATIVE - POSITIVE + POSITIVE + +Fig. 1 O-l 4 The orthotolidine method for occult blood

Measuring the pH of a stool and testing for lactose 1 10.12his gut (stomach and intestine) the blood may not beseen. This is because it is digested and mixed up with thestool. Bleeding into the gut that cannot be seen is calledoccult bleeding. Occult means hidden. When we test foroccult blood we test for hidden blood that cannot be seenby simply looking at the stool.Bleeding is not always occult. When a patient bleedsfrom near the bottom of his gut, the blood is not digested,and bright red blood is seen in the stool. Also, if there ismuch bleeding high up in the gut, the blood may not bedigested before it leaves the body. Here too, bright redblood may be seen in the stool. Sometimes there is heavybleeding high up in the gut and the blood is partlydigested before it leaves the body. This half-digestedblood is black. A black stool like this is called a melaenastool.METHODTESTING FOR OCCULT BLOOD IN THE STOOL. FIGURE lo-141. Pour 5 ml of glacial acetic acid into a test tube. Fivemillilitres is two finger’s breadth deep.2. Add to it 200 mg of ortho-tolidine-barium peroxidemixtureisee Section 3.351. The best way to measure thisout is to keep the mixture in a bottle with a little tinscoop pushed into its cork. When you first make thescoop, weigh out 200 mg of mixture and see what itlooks like on the scoop. Remember what this looks likeand add the same amount next time. Dissolve the orthotolidioe-bariumperoxide mbrture in the glacial aceticacid. The mixture will go green.3. Put a piece of filter paper in the sink, or on to a tileor dish. Smear some stool on to it.4. Pour some of the glacial acetic acid mixtureover it.5. If there is no colour change in 30 seconds, the testis negative (-1. The paper will of course stay pale greenishwhere it was stained by the reagent.If the stool goes green after 30 seconds, the test isweakly positive (+).If the stool goes deeply blue-green within 15 seconds,the test is strongly positive (++I. It is not possible tojudge ‘+++’ and ‘++++’ with this method.Ortho-tolidine is under suspicion as a dangerouschemical, for it is thought that, if small quantities aretaken into the body over a long time, they may causecancer. In some countries its use has been stopped bylaw..However, it is not certain that the small amountsused m a medical laboratory have ever caused this disease,and there is no other suitable test for occult blood.This test has therefore been described here for use inthose countries where ortho-tolidine may still be used.However, don’t spill it round the laboratory, or it mayget into the dust, into the air, and so into you. Don’tget it on to your hands, and wash them after you havebeen using it.10.12 Measuring the pH of a stool and testing forlactoseIn Section 1.7 you read how we use pH to measure theacidity or alkalinity of a solution. pH 1, you will remember,was very acid, and pH 14 was very alkaline. pH 7 isin the middle; it is neutral and is neither acid or alkaline.Pure water has a pH of 7. The pH of a stool is easilymeasured and is a useful way ‘of telling if diarrhoea isprobably due to a bacterial infection or to lactoseintolerance. Lactose is a sugar which is found in milk.Used like this, intolerance means being unable to digestand absorb something. All the lactose in the food ofhealthy people is digested in the gut by an enzyme calledlactase and is absorbed into the body. But in somepatients, especially those with kwashiorkor (see below),there is too little lactase in the gut and lactose is notproperly digested and absorbed. It stays in the gut and ismade into lactic acid by the normal bacteria of the gut.This lactic acid causes diarrhoea with an acid stoolwhich has a pH of less than 6. In a bacterial diarrhoea(such as a shigella infection-see Section 10.8) the stoolis alkaline with a pH of more than 7. By testing the pH ofa patie.nt’s stool we can therefore tell if his diarrhoea ismore likely to be due to intolerance to lactose (or someother sugar) or to bacterial infection. There are of courseother causes of diarrhoea, such as the diarrhoeas causedby Giurdia and amoebae, where the pH of the stool doesnot help us.When children do not get enough protein or enoughjoule-containing (energy-containing) foods, they do notgrow properly and become sick. They are said to haveprotein joule malnutrition or PJM. A child with severePJM suffers either from a disease called kwashiorkor, orfrom another disease called nutritional marasmus. Insome countries these are both common and importantdiseases which kill many children. PJM can be preventedby proper feeding, especially feeding with protein. Acommonly used protein food is dried skim milk, which isabout half protein and half lactose. This gives somechildren diarrhoea. If it gives a child diarrhoea with anacid stool of pH less than 6, he probably has lactasedeficiency. The way to get over this is to ask his motherto add a little dried skim milk to all his food, andespecially to his porridge. Given in this way it is lesslikely to give him diarrhoea. Another and usually lesseasy way is to give the child another protein food, whichdoes not contain lactose.METHODMEASURING THE pH OF A STOOLlake a piece of universal indicator test paper and dipone end of it into the stool.Where the paper is covered by stool you will not beFootnote. The energy in foods used to be measured in calories. Themodern way is to measure it in joules which are part of the SI orInternational System of units. There are 4. I8 joules in a calorie.

able to see its colour, but the water in the stool will soakinto (move into) the paper and change its colour. Universalindicator test paper is a dirty yellow colour. If thestool is alkaline, as in a bacterial diarrhoea. the paperwill go green or even blue (pH 7 and above). If the stoolis acid, as in lactose intolerance, the paper will go brown(pH 5). A pH of 6 or less in the stool of a child takingmilk in his food usually indicates lactase deficiency.These are the colours for the universal indicator testpapers listed in Section 13.10. Other kinds of universalindicator test paper may change colour in differentways.It is also possible to test for lactose directly by testingthe stool with a test tablet that is usually used for testingthe urine for sugar. This is the ‘Clinitest’ tablet (AME).This is a much better test for lactose intolerance than thepH of the stool.METHODTESTING FOR LACTOSE IN THE STOOL WITH A ‘CLINITESTTABLETCollect the stool by putting a piece of polythene sheetinside the baby’s napkin. This is important because thelactose is in the watery part of a stool which is absorbedby a cloth napkin.Make a short, wide Pasteur pipette. Put about half aninch of the diarrhoeal stool in a test tube. Add twice asmuch water. Mix the stool and the water by drawingit in and.out of the pipette. Add fifteen drops of this stooland water mixture to a second test tube. Add a‘Clinitest’ tablet. If there is lactose in the stool, the liquidin the tube will go green, yellow, or brown, just as if thetablet were being used to test urine for sugar. If a childis taking milk and his stool goes yellow or brown by thistest (more than O-5% lactose), he probably has lactoseintolerance.10.13 When to examine the stoolsNow that you know what abnormal things can befound in stools, you can understand when to examinethem.Look at the stools of all patients with diarrhoea thatdoes not get better rapidly, especially if there is blood inthe stools. You may find amoebae or the ova ofSchistosoma mansoni or Giardia lamblia. You may alsobe able to tell if the patient has an amoebic or bacillaryexudate.Look at the stools of all anaemic patients to see if youcan find hookworm ova. As you read in Section 7.6,hookworms are a very common cause of hypochromicanaemia. Look for occult blood in the stools of allpatients with a hypochromic anaemia and no obviouscause for bleeding, such as many hookworms in the gutor heavy bleeding from the womb (menorrhagia). Thepatient may be bleeding into his gut from some othercause, such as a peptic (stomach) ulcer.Look at the stools for ova in any patient who has hadabdominal (stomach) pains some days or weeks. Manyworms cause abdominal pains, and many patients haveabdominal pain; so you will often have to examine stoolsfor this reason.If children are thin and not growing well, look for ovain their stools. Sometimes their thinness is caused byworms. More often it is because they do not get enoughgood food to eat.QUESTIONS1. Which worms can be seen in the stool? Describeeach of the species of worm that you might see.2. What is a standard faecal smear? How would youmake one?3. In what way do the ova of S. mansoni differ fromthe ova of S. haematobium?4. How would you look for the ova of Enterobiusvermicularis?5. How would you distinguish Entamoeba histolvticafrom Entamoeba coli?6. What is meant by a bacillary and an amoebic exudate?7. What flagellates do you know? How do you lookfor them?8. How may blood be found in the stool?9. What is the importance of lactose in a diarrhoeastool?10. What are the advantages of the formal-ether concentrationmethod? How is it done?

,-?‘,C ,:-. 1,I”.&‘.;-,__,-_ i _._, ,-. ~.;-y “C‘~“ ,,~.. .)1,;:_“’ .‘.11 1 Some Other SpecimensSPUTUM11 .l AAFB and the Ziehl-Neelsen methodThere is an important genus (tribe) of rod-shaped bacteriaor bacilli called the mycobacteria. Mycobacteriumtuberculosis causes tuberculosis or ‘TB’, andMycobacterium leprae causes leprosy. They are oftencalled tubercle bacilli and leprosy bacilli. Tuberculosis isusually a disease of the lungs, and tubercle bacilli canoften be found in the sputum. Leprosy is usually adisease of the skin and nerves, so we look for leprosybacilli in smears made from a small cut in the skin.Even though tuberculosis and leprosy harm differentparts of the .body they are like one another in severalways. In both of them the infection may start when thepatient is a child. Both are usually chronic diseases iastingmany years which cause severe disability if they arebadly treated, or if treatment is started too late. Bydisability we mean that the patient is unable to lead anactive life and particularly that he is unable to work.Patients seldom die from leprosy, but they often die fromtuberculosis. Both diseases can usually be cured ifpatients are diagnosed early and if they take their drugsregularly and for long enough. In leprosy infectionalways takes place from one person to another. This isalso the usual way in tuberculosis, but there is a lesscommon kind of tuberculosis called bovine tuberculosisin which infection is caused by drinkinginfected milk from an infected cow. Bovine means froma cow.Both Myco. tuberculosis and Myco. leprae can bestained by the Ziehl-Neelsen method, which is namedafter the two people who first invented it. This methoduses a solution of a bright red stain called basic fuchsinin a mixture of water, phenol, and spirit. Phenol used tobe called carbolic acid, which explains why the mixtureis called ‘strong carbol fuchsin’. Dilute carbol fuchsin isused for Gram’s method. The specimen is spread on aslide to make a thin film. This is then fixed by beingpassed quickly through a flame, after which it is coveredwith strong carbol fuchsin. The carbol fuchsin is thengently heated for 5 minutes. All bacteria stain a deep red,so do the cells in the specimen. The hot stain is nextpoured off, and the slide is covered with a mixture of acidand alcohol called acid-alcohol. This washes away thered stain from the cells in the specimen as well as frommost of the bacteria. It is said to decolorize them. But theacid-alcohol does not wash the red colour away frommycobacteria, which stay a deep red when everythingelse on’ the slide goes pale. Because mycobacteria holdon to the red stain in this way they are said to be ‘Acidand Alcohol Fast’. When used like this the word ‘fast’means ‘able to hold on to a stain’. When we look atsputum that has been stained by the Ziehl-Neelsenmethod we may see bacteria that have stayed red whenacid-alcohol has been poured on them. We see Acidand Alcohol Fast Bacilli or AAFB. We thus reportAAFB present or absent iu the specimen. Myco. lepraeis slightly less acid fast than Myco. tuberculosis. Filmsfrom leprosy patients are therefore stained for a longertime with carbol fuchsin than films from patientswith tuberculosis, and are decolorized with a weakeracid-alcohol.A film is easier to look at if the cells in it are coloured.Red mycobacteria are also easier to see when the cells inthe film are stained a different colour. But acid-alcoholwashes the red fuchsin away from the cells of the sputumor skin smear which become pale, colourless and hard tosee. If therefore the cells are going to be coloured, theymust be stained again. Either malachite green ormethylene blue can be used. Stains like this, which areused to stain the less important parts of film (the cellsrather than the mycobacteria), are called counterstains. Ifyou use malachite green as a counterstain in the Ziehl-Neelsen method, you will see red mycobacteria andgreen cells.We shall describe two ways of doing the Ziehl-Neelsen method. One is the ‘hot method’ which has beenused for many years. The other is the ‘cold method’which is newer and uses only two solutions (instead ofthree with the hot method) and no heat. With most stainsfilms are best stained one at a time, but with the Ziehl-Neelsen method several films can easily be stained together.The strong carbol fuchsin stain for the twomethods is slightly different and is described in Sections3.22b and 3.23. The stronger acid meant for tuberclebacilli can be used for leprosy smears, but it is better notto and to use a weaker acid instead.

11 1 Some Other SpecimensMETHODZIEHL-NEELSENSTAINA. THE HOT METHOD. FIGURE 11-l1. Look at the sputum carefully. If it is in a widepolypot it will be easier to look at than if it is in a narrowbottle. Find a piece of sputum which is thick, yellow, andpurulent (pus-like). A piece of sputum like this will bemore likely to have mycobacteria in it than will otherparts of the specimen. If the specimen is frothy andwatery, it is probably only saliva from the mouth, and itis better to ask for another one.2. Flame your loop well. When it is cool take hold of apurulent piece of sputum. When the sputum is verysticky and tough, you may find it easier to use twoloops.3. The purulent piece of the specimen chosen forstaining must be small--a little smaller than the head ofa match.4. Spread or smear the small piece of sputum on aclean slide. Spread it very thinly. As you spread thesputum it will dry and stick to the slide. By the time ithas been spread out completely, the film will probablybe very nearly dry. Let the film dry completely. FLAMEYOUR LOOP OR LOOPS AGAIN BEFORE LAYINGTHEM ON THE BENCH. If there is a lump of sputum onthe end of your loop, get it off before flaming by dippingthe end of the loop in a little dish of pure lysol which youshould keep on your bench. This will stop mycobacteriaspitting out of the flame on to the bench.5. With the film towards the flame hold the slide in aBunsen burner or spirit lamp for a very short time--onlya second or two. This kills the mycobacteria and thecells and is said to ‘fix’ the film (see Section 4.10).6. Place the slide on a staining rack and move therods of the rack so that the slide is quite flat or horizontal.The film can then be covered with stain more easily.7. Pour on strong carbol fuchsin for the hot Ziehl-Neelsen method (Section 3.22b). not dilute carbol fuchsin,until the slide will hold no more-that is, until stainjust does not run off the slide. The whole of the slidemust be covered with stain. If carbol fuchsin starts todry up in the bottle and a yellow scum comes on thesurface, add a little spirit to the stain. This will dissolvethe scum.6. Heat the slide until the stain just starts to steam,but no more. THE STAIN MUST NOT BOIL. If the stainboils or dries, there will be lumps of solid fuchsin all overthe finished slide. Use a Bunsen burner, a spirit lamp, ora swab soaked in spirit. A cotton wool swab soon burns,and a better swab can be made from asbestos wool onthe end of a piece of wire, the other end of which isstuck into a cork (9). Asbestos is a soft substance likeordinary cotton or wool, but it is not burnt by a flame. Anasbestos swab of this kind is often the easiest thing touse. When it is not being used, keep it in a jar of spirit(IO).When the flame is taken away the hot stain on a slidewill go on steaming for about a minute and then stop.Warm it again carefully a second time until steamingstarts once more.11. When staining for Myco. tuberculosis leave thewarm stain on the slide for 5 minutes. When staining forMyco. liprae leave it on for 10 minutes. Use a pair offorceps to tip the stain off the slide and keep it awayfrom your fingers. The film will now be stained a deepred.12. Wash the slide in water.13. Put the slide back in the rack and cover it withacid-alcohol. Use 3% acid-alcohol for Myco.tuberculosis and 1% for Myco. ieprae. The acid-alcoholwill take the stain out of the film which will becomemuch paler.14. Wash the film in water rapidly once again. After3% acid-alcohol it will be almost colourless, but after1% acid-alcohol it should still be a very faint pink colour.15. Lay the slide on the rack and cover it with malachitegreen. Only a few drops will be needed-justenough to cover the film. Leave this on for about 15seconds. Use 0.3% methylene blue if you have no malachitegreen.16. Wash the slide with water for the third time.17. Leave the slide to dry in a rack.6. THE COLD METHODMake your films, fix them in a flame and put them onthe staining rack just as in the hot method.Cover the films with strong carbol fuchsin for the coldZiehl-Neelsen method (Section 3.231. SHAKE THEBOTTLE before pouring on the stain. Pour the stain onto the slides through a filter paper held in a small funnel.Keep the funnel and the paper for this stain only. Thereis no need to wash the funnel each time you use it. Thestain need not cover the whole slide as is so importantwith the hot method.Leave the stain on the slides for 10 minutes for Myco.tuberculosis and for 30 minutes for Myco. leprae.Wash the slides well with water.Tip off the water.Cover the slides with ‘Methylene-blue-acid-alcohol’(see Section 3.351 and leave them for 3 minutes exactly.Use 8% acid for Myco. tuberculosis and 1% for Myco.leprae.Wash the slides well with water. Leave them to dry ina drying rack.If you have many slides to stain, label them with adiamond pencil so that they do not get mixed up.Another way of labelling a slide is to label it in greasepencil on the underneath where the writing will not getwashed awav by the stain. Keep the stains in wash bottles.You can easily stain many slides at once, but be carefulnot to have so many that you should be washing the firstslides before you have finished staining the last ones.Look at a jilm with a low power objective for a goodplace to search with an oil immersion objective. Withpractice you will soon learn how to find a good place,

find a purulent(pus like) pieceof the specimen: L,F.i/2flame+ ; \ loop3pr-// flame4Y\ 1\\FBunlen burner 1 Bunsen burner L film towards -the flamethis is a specimenof sputum in aplastic ‘Polypot’this is strong carbolfuchsinfor the hotZiehl-Neelsen methodmake sure the slide is quiteflat or horizontalY6 .Aspread the specimenout very thin on the /slideheat the slideuntil the steamjust rises butno more DON’Tlet the stain boil :pour on strongheat the slide illi Icarbol fuchsinonce again during _until the slideis COVEREDthe five minutesof staininawith stainAgreen for a fewseconds only-16 w,'14, \ Ileave the film to!. Ly:‘,-B drv in a rack,/OIL IMthe film should platesMERSIONVIEslide rack’/u!the cells are green ’1115 IIIyLVUclLLGilla arc “alylll -red thin rodsFig. 1 l-l Ziehl-Neelsen’s method

11 1 Some Other Specimenswhich must not be too thick, or too thin. When methyleneblue has been used as a counterstain, as in the coldmethod, you will see deep red bacilli and blue cells.When malachite green has been used, the cells will begreen. Mycobacteria often have granules on them so thatthey look like a string of beads. Mycobacteria may alsolie in groups of several bacilli together.If there are many bacilli in the sputum you will findthem quickly. But if there are only a few bacilli in thesputum you may have to search for a long time. Howlong should you search before stopping and saying that aslide is negative? This will depend upon how many slidesthere are and how much time you have. If possible searcha slide for 10 minutes before reporting it as negative, anddon’t look at less than about 100 fields.It is usually easy to decide if something you see is anacid-fast bacillus or if it is not. If you see something youare not sure about, don’t report it as positive, but look atother parts of the film to see if you can find bacilli youare sure about. If you are still doubtful, even after youhave searched the slide very carefully, report ‘AAFBdoubtful’, and ask for another specimen of sputum fromthe same patient.11.2 Preventing false positive reportsBecause the right diagnosis of tuberculosis or leprosy isso important to the patient, you must not make falsepositive reports. A false (not true) positive report is areport which is given as positive when it should be negative.A false positive report will cause a patient to betreated for tuberculosis or leprosy when he is reallysuffering from some other disease. He will therefore begiven the wrong treatment and will not be cured. How dofalse positives happen, and how can they be pre&nted?You have already read about one reason for false positivereports-saying something is an acid-fast bacillus whenyou are not sure about it. Such a report should rially bedoubtful (t).A common cause of false positive reports is bacilligetting from a positive film on to a negative one. Thismay happen if a positive film is badly washed, and thenused for another film. Stained mycobacteria from the firstfilm may be left on the slide and cause the second specimento be falsely reported as positive. The best way toprevent this is to break evecv positive AAFB slide so thatit cannot be used again. It is much better to waste a fewslides than to run the risk of false positive reports. If youhave very few slides and must use every one, keep thosefrom positive Ziehl-Neelsen films and use them for othermethods. Try to use new slides for leprosy smears, especiallyif yo\i are doing the bacteriological and morphologicalindi+s.Another icause of false positive reports is to carrybacilli from one slide to another with the rod of an oilbottle. If your oil bottle has a rod, don’t touch the surfaceof the slide with it. Instead, let a drop of oil fall from therod on to the slide (see Picture 22, FIGURE 7-7). Don’tuse jars of stain because bacilli may go into the stain andstick to negative films. These films will then be falselyreported positive.11.3 Harmless mycobacteriaUntil now you have only read about two kinds of mycobacteria,Myco. leprae and Myco. tuberculosis. But thereare other species of mycobacteria, and most of them donot cause disease. They live on the skin, in water, and inthe soil. These harmless mycobacteria are acid fast likeMyco. tuberculosis and MJTO. leprae, but some of themare not alcohol fast. They are therefore decolorized(made colourless) by the alcohol in acid-alcohol and donot stain red by the Ziehl-Neelsen method. Even so,when we stain the sputum by the Ziehl-Neelsen method,it is possible that the bacilli we see are not Myco.tuberculosis but some harmless species from somewhereelse. We thus always report ‘AAFB present’ and not‘Myco. tuberculosis or Myco. leprae present’. To tell ifAAFB are really Myco. tuberculosis we have to growthem and see what they look like. We cannot do this inour laboratory. Even so, we almost always treat patientsas if the AAFB found in their sputa by the Ziehl-Neelsenmethod were certainly Myco. tuberculosis. Harmlessmycobacteria are uncommon. If more than one specimenis found with AAFB, it is almost certain that the patienthas tuberculosis.11 Aa Finding cases of tuberculosisPatients with AAFB in their sputum are said to besputum positive. Those without AAFB are said to besputum negative. When you find AAFB in the sputum ofa patient this means that he has tuberculosis of his lungs-pulmonary tuberculosis. It also means that his pulmonarytuberculosis is both ‘active’ and ‘open’. By‘active’ we mean that mycobacteria are growing, thatthey are destroying the patient’s lungs and that he isgetting more ill. By ‘open’ we mean that mycobacteriaare getting out of the patient and may infect someoneelse. Mycobacteria from someone with open pulmonarytuberculosis can spread in the air in little drops ofsputum when he coughs or spits. In this way they canspread from a patient to a healthy person, who may thenget tuberculosis and become ill. This is the most commonway in which tuberculosis spreads from one person toanother. If a patient has tuberculosis but has not gotAAFB in his sputum, he is probably not infectious and issaid to be a ‘closed’ case.If tuberculosis is to stop spreading in a town or village,all cases of open tuberculosis must be found andtreated. An important way to find cases of open tuberculosisis to remember that any patient may have tuberculosiswho has a cough which has lasted more than onemonth and is coughing up sputum. A patient with acough who is producing (coughing up) sputum is said tohave a productive cough. If there is blood in a patient’ssputum, he is even more likely to have tuberculosis.Coughs lasting less than one month are usually due to

Examining the sputum for helminth ova 1 11.4bmild infections of other kinds and are not important. Aswell as having a productive cough which has lasted morethan one month, a patient with tuberculosis is often thin(he has lost weight) and may feel unwell. You will findAAFB in the sputum of about one patient in twenty witha cough, so you must look carefully to find cases oftuberculosis.If a patient has tuberculosis of his lungs very badly,there will be many AAFB in his sputum, and it will beeasier to find them than when he only has it mildly.Milder cases of tuberculosis do not cough up AAFB inevery specimen of sputum. so it is necessary to examineseveral specimens to have a fair chance of finding AAFB.Always examine three, and better six specimens, beforetelling a patient who has had a cough for over a monththat he has not got tuberculosis. One or two negativesputa are not enough.Patients with tuberculosis often spread it to theirfamilies and to the people they work with. We call thesepeople close to the patient his contacts. If the contacts ofa tuberculosis patient have coughs and are coughing upsputum, this sputum must be examined. These contactsare usually traced (looked for) by health assistants’ ornurses, and not by laboratory assistants. But laboratoryassistants must stain the sputum from these contacts andlook for AAFB. Looking at the sputum of tuberculosispatients and their contacts is one of the most usefulthings a laboratory assistant can do. This is becausetuberculosis will go on spreading until these cases can befound, shown to have AAFB in their sputum and treated.Once a patient is sputum negative, he will no longer be adanger to other people, and tuberculosis will stop spreading.Children are especially likely to catch tuberculosiswhen there is a case of open tuberculosis in the family.But children swallow their sputum and seldom cough itup. Because of this, tuberculosis in children has thereforeto be diagnosed in a different way.If sputum is to be obtained from a child, it has usuallyto be sucked up and washed out of his stomach witha rubber tube through his mouth. This is how it isdone.METHODGASTRIC WASHINGS IN YOUNG CHILDRENAsk the child to swallow the end of a rubber stomachtube before he has had anything to eat in the morning.Suck up any liquid there is in his stomach. Put this inone of the universal containers containing buffer that aredescribed in Section 3.20a.If there is not enough fluid to fill the containrtr halffull, put a little water into the stomach through thesyringe. Suck this out and put it into the universalcontainer.Send the specimens to a central laboratory as soon aspossible. The central laboratory will try to grow Myco.tuberculosis.TreatmentTuberculosis is usually cheap and easy to treat. Butpatients must understand that they should take theirtreatment regularly for at least a year and sometimes for2 years. They are often given an injection of a drug calledstreptomycin for about 6 weeks. They are also usuallygiven tablets of a drug called INH for at least a year, andwith it either a drug called thiacetazone (also called TBl), or another drug called PAS. Tests for some of thesedrugs in the urine are given in Section 8.9.11.4b Examining the sputum for helminth ovaSputum can be examined for many micro-organisms.Much the most important is the Ziehl-Neelsen methodfor AAFB that has just been described. The only otherone that we shall describe here is a concentration methodfor helminth ova, and particularly for the ova of thetrematode Paragonimus westermani that is commonlyfound in some parts of the world. This trematode lives inthe lungs and its ova are coughed up in the sputum. Youmay be able to find them by looking at a saline smear ofthe sputum under a coverslip. Occasionally the ova ofother helminths may be found by the same method. Aswith Myco. tuberculosis, look at the thick yellow orblood-stained parts of the sputum. If you cannot find theova in a saline smear look for them using this concentrationmethod.METHODA CONCENTRATION METHOD FOR HELMINTH OVA IN THESPUTUMMix the sputum well with about a quarrer of itsvolume of 20% sodium hydroxide (see Section 3.42b).Let the mixture stand for about 10 minutes.Centrifuge and examine the deposit under a coverslipfor helminth ova-see Figure 10-9.PUS11.5 Gram’s methodThis is one of the oldest stains for bacteria. It is also oneof the best. A film is made from the specimen. This filmis then quickly fixed in the heat of a flame and stainedwith a violet stain called crystal violet (violet is a specialkind of blue). After this a few drops of Lugol’s iodinesolution are dropped on to the film. Spirit is next pouredover the film, which is then washed in water and counterstained(see Section Il. 1) with dilute carbol fuchsin.All bacteria and all cells stain a deep violet with thecrystal violet followed by the iodine. Some bacteria staythis deep blue colour when the spirit is poured overthem. Bacteria which stay a deep violet are said to beGram positive. Spirit washes away the violet colour fromall cells and from many bacteria and makes them colourlessagain. Bacteria which are decolorized by spirit are

11 1 S&A O&r S$&imenssaid to be Gram negative. These colourless bacteria andcells are then stained red with the dilute carbol fuchsincounterstain.- In the Ziehl-Neelsen method a counterstain(malachite green or methylene blue) is used to stain cellsor bacteria that have been decolorized in acid-alcohol. Acounterstain is also used in Gram’s method, but this timeit is the red stain, dilute carbol fuchsin. Gram positivebacteria are therefore violet, because they hold on to theviolet stain when treated with spirit. Gram negative bacteriaare red, because they are decolorized by the spiritand are then stained again with the red carbol fuchsin.There are very many species of bacteria. About half ofthem are Gram positive and half Gram negative. Gram’sstain can thus be used to divide bacteria into two largegroups of about equal size, the Gram positive species andthe Gram negative ones. In this Gram’s method and theZiehl-Neelsen method differ from one another. With theZiehl-Neelsen method only one genus, the mycobacteria,is acid fast. All the many other genera are non-acid fast.The Ziehl-Neelsen method thus divides bacteria intotwo groups of very unequal size. There is a small groupof acid-fast bacteria and a very large group of non-acidfastbacteria. The words ‘Ziehl-Neelsen positive’ and‘Ziehl-Neelsen negative’ might have been used, butinstead we use the words ‘acid fast’ and ‘non-acid fast’,METHODGRAM’S STAIN. FIGURE 11-21. Take a loopful of pus (2) or the centrifuged depositfrom a specimen of CSF 131. Smear it on a clean slide(4). Make a thin film which will dry as it is spread on aslide.5. Fix the film in a flame for a moment, making surethat the film faces the flame.6. Hold the slide in your hand over a sink, a dish or abucket. Don’t put it on the rack as is done with theZiehl-Neelsen method. Drop a few drops of crystalviolet stain over the film (Section 3.261. Cover the filmwith the crystal violet, and take care to keep the stainaway from your fingers.7. Pour a few drops of Lugol’s iodine (Section 3.32)over the film.6. Make sure you have plenty of water ready. If youhave a tap. turn it on. Pour spirit over the film until theblue stain just stops running from it but no longer. Thiswill take longer in some slides than in others, but it isusually about 6 seconds.9. Immediately the stain stops running from the film,wash it in water. If there is no running water, pour a cupof water over the slide.10. Still holding the slide over the sink, pour a fewdrops of dilute carbol fuchsin over the film.11. Without waiting, rapidly wash the film in water.12. Leave the slide to dry in a rack.13. Using a low power objective find a good part ofthe film to look at with an oil immersion objective.Gram positive bacteria will be a deep violet. Gramnegative bacteria and all cells will be red.*If the cells andespecially their nuclei are still violet, the film has notbeen treated for long enough with spirit. Don’t continueto pour spirit over a film after the violet stain has stoppedrunning from it. If you do, you may decolorize it toomuch, and even some Gram positive bacteria may bedecolorized, and be stained red instead of violet in thefinished film.. Gram’s method can be used for specimens of severalkinds. Its use in meningitis is described in Section 9.16.It is also very useful for diagnosing gonorrhoea.11.6 Urethral smears for gonococciA venereal disease is a disease which spreads from oneperson to another through sex. One of the venerealdiseases is called gonorrhoea. It is caused by bacteriacalled Neisseria gonorrhoeae, N. gonorrhoeae, or gonococci.When a man has gonorrhoea pus comes from hisurethra. This pus is called a urethral discharge. The urethrais the tube inside the penis (a man’s sex organ)through which urine flows. This pus can be stained withGram’s stain to show Neisseria gonorrhoeae.METHODURETHRALSMEARSMake films of pus by taking a clean slide and holdingit against the drop of pus at the end of the patient’s urethra.Spread the pus to make a thin film. Wave the filmdry and fix it quickly in a flame. Write the patient’s nameon the slide and stain it by Gram’s method.Neisseria gonorrhoeae is a species of Gram negativediplococcus very like Neisseria meningitidis (see Section9.16). Diplococci are cocci which are seen in pairs (dimeans two)-that is, two cocci are usually found closetogether. The Neisseria are shaped like a bean, and thelong sides of a pair of cocci touch one another as shownin Picture E at the bottom of FIGURE 1 l-2. In a urethraldischarge from a patient with gonorrhoea gonococciare usually seen inside pus cells or intracellularly (intrameans inside), as in Pictures A to D at the bottom ofFIGURE 1 l-2. If Gram negative intracellular diplococciare seen in a patient’s urethral smear he probably hasgonorrhoea. Just as we report ‘AAFB seen’ (not Mycg.tuberculosis seen), so we report ‘Gram negative intracellulardiplococci seen’ (not N. gonorrhoeae seen). We dothis because we cannot be quite certain without specialmethods that the Gram negative intracellular diplococciwe are seeing are really N. gonorrhoeae. Harmlessspecies of Neisseria are found in urethral discharges, butthey are not usually found intracellularly inside pus cells.When looking at a urethral smear, always look for Gramnegative diplococci inside pus cells and take no notice ofGram negative diplococci outside pus cells. These maybe N. gonorrhoeae or they may be harmless species. Send

this is the centrifuged depositfrom a specimen of CSFspread out the pus or CSF depositto make a thin even filmcrystalviolet9flame the slidevery quickly withthe film_.towardsthe flamedropper from the‘Polvstop’ bottleof Lugol’s iodinefor a few secondsthe film UNTILTHE STAIN CEASESTO RUN BUT NOdilute carbol fuchsin 11OIL IMMERSIONVIEWleave the slideNEISSERIA AND PUS CELLSextracellular diplococci intracellular diplococcioutside pus cells GRAM POSITIVE BACTERIA STAIN VIOLETGRAM NEGATIVE BACTERIA STAIN REDFig. 1 l-2Gram’s methodthis picture shows two bean shaped Neisseria lyingclose to one another and drawn very large

~; -I ,_ i _ ,11 1 Som*~Other Specimensout positive reports like this ‘Gram negative intracellulardiplococci seen’.Women may also have gonorrhoea, but it is less easyto diagnose in them. This is partly because a vaginaldischarge in a woman (the vagina is the passage throughwhich a child is born) is less easy to see than a urethraldischarge in a man. Also, there are often so many diierentbacteria in the vagina that it is hard to lind Neisseria.11.7 Some less common uses of Gram’s methodIn our laboratory the main use of Gram’s method is tostain the CSF and diagnose gonorrhoea. There arehowever some other less common uses for it.If pus from an abscess is stained with Gram’s stain,Gram positive cocci may be seen. An abscess is a placein the body containinng pus. These cocci have beenshown in Picture 13, FIGURE 11-2. Cocci are often seenin a chain like a necklace of beads or close to one anotherin a group or clump. Two main genera of Gram positivecocci cause disease in man-streptococci and staphylococci-butit is difficult to tell one from the otherwithout culturing (growing) them.There is an uncommon disease called anthrax whichmen catch from cows, sheep. and’other animals. It iscaused by a big Gram positive bacillus (Bacillusanthrucis). These bacilli can be found in Auid from theanthrax lesions (diseased places) in the skin.There is another uncommon disease called plague. Thelymph nodes in the groin get big, and pus forms abscessesinside them. If some of this pus is taken fromthem with a needle and stained by Gram’s method, smallfat Gram negative bacilli can be seen. These bacilli oftenstain more strongly at their ends.. They are calledYersinia pestis.VAGINALDISCHARGES11.8 Looking for Trichomonas vaginalisThis is a protozoon which lives in the vagina of womenand sometimes in the urethra of men. In women it oftencauses a vaginal discharge, and in men it may cause aurethral discharge. Any moving protozoon you find inthe discharge from the vagina or urethra is almost sure tobe Trichomonas vaginalis.METHDDLOOKING FOR TRICHOMONAS VAGINALISUsing a Pasteur pipette put a drop of fluid from thevagina on to a slide. Cover the drop of fluid with acoverslip and look for moving protozoa.When looking for T. vaginalis in men put a drop of theurethral discharge on a slide. Look at it wet under Bcoverslip and examine it for moving prdozoa.Look at these slides quickly before the protozoa dieand stop moving. It is much better to look at thesespecimens in the outpatient department than to sendthem to the laboratory. If specimens have to be sent to alaboratory, take a few drops of fluid into a Pasteurpipette and put the pipettte into a test tube. Take it tothe laboratory quickly and look at it immediately.GASTRICJUICE11.9 Testing gastric juice for free acidIn Section 7.19 you read about macrocytic anaemias. Inone kind of macrocytic anaemia called pemicisusanaemia the acid in the stomach is very weak. We saythat there is ‘no free acid in the gastric juice’ (the gastricjuice is the liquid made by the stomach). A specimen ofgastric juice should be taken in the ward on the instructionof a doctor. This is what he will ask the nurses in theward to do.METHODFREE ACID IN THE GASTRIC JUICEGive a fasting patient 100 mg of mepyramine(Anthisan) intramuscularly.‘Half an hour later give himO-04 mg/kg of histamine subcutaneously. Half an hourafter that ask the patient to swallow the end of a rubberstomach tube. Suck up some of the juice in his stomach.Put a piece of universal indicator test paper into thejuice (see Section 1.8). If there is ‘free acid’ in the juicethe universal indicator paper will show a pH of less than4. Mot all universal indicator papers change colour in thesame way, but the paper described in Section 13.10should become a dsep orange-red colour. Congo redpaper is a better test of free acid. It goes blue if there isfree acid in the gastric juice.SEMINALFLUID11 .lO Examining the seminal fluidHusbands and wives are often worried because they wantchildren and do not have them. Sometimes this is becauseof the wife, and sometimes it is because of the husband.One way of finding out whether the husband or the wifeis the cause is to look at the husband’s seminal fluid (hisseed). If there are not enough spermatozoa or ‘sperms’(the special highly motile male cells) in the husband’sseminal fluid, the lack of children is probably due to him.Spermatozoa have been drawn in Picture 18, FIGURE8- 10. There are several methods of examining theseminal fluid, but we shall only consider two. One is tocount the total number of spermatozoa in each millilitreof tluid. They are counted in a counting chamber in thesame kind of way as white cells in the blood or CSF. Theother method is toexamine the motility or movement ofthespermatozoa. This is very easy. A drop of seminal fluid islooked at under a coverslip with a high power objective.

Classifying leprosy 1 11 .l laIn normal seminal fluid motile spermatozoa can be seenmoving about very actively.METHOOEXAMINING THE SEMINAL FLUIDTHE TOTAL SPERM COUNTGive the patient a clean, dry container and explain tohim how he is to bring a specimen of seminal fluid assoon as he has passed it. Seminal fluid is a liquid when itis passed. It soon clots and then goes liquid again. Measure10 ml of water into a universal container. MeasureO-05 ml of seminal fluid in a blood pipette. Add it to thewater. Mix well. Using a Pasteur pipette till a Neubauercounting chamber with the diluted seminal fluid suspension.Count all the complete spermatozoa you see intwo blocks of 16 squares (O-1 mm). By complete wemean those with heads and tails. Multiply the numberyou find by a million (add 000,000 to it). This will giveyou the number of spermatozoa in 1 ml of seminal fluid.A normal man has more than 40.000.000 sperms ineach millilitre of seminal fluid. So you should find atleast forty sperms in two blocks of sixteen smallsquares. Report the number of spermatozoa you find.MOTILITYPut a dropful of very fresh seminal fluid on a slide. Puta coverslip on it and look at it with a high-poweredobjective with the condenser moved down a little. Innormal seminal fluid at least 80% of the spermatozoawill be actively moving.If there are no spermatozoa the man is said to sufferfrom azoospermia. Azoospermia is a common cause of aman’s failure to have children. Sometimes spermatozoacan be seen but there are fewer than there should be.There may perhaps be only 5 million per ml instead of40 million.in Picture A, who are able to live with an infectious oropen case of leprosy for many years and yet never getleprosy. Because they are so good at fighting leprosythey are said to have a high resistance to it.A few people are unable to fight the bacilli well; so thebacilli win the fight and are able to multiply and causesevere disease. These people lose the fight against theleprosy bacilli because they have no resistance toleprosy. The severe kind of leprosy they get is calledlepromatous leprosy (Picture D).Between the many people with a high resistance toleprosy and the few people with a very low resistance to itthere are others with some resistance, but not enough forthem to win the fight completely. These people are onlyfairly good at fighting leprosy and get a mild kind of thedisease called tuberculoid leprosy (Picture B) or a moreserious kind called borderline leprosy (Picture C).There are thus several kinds of leprosy which dependmostly on how much resistance a patient has to the bacilli.In each kind of leprosy the patient may have any of severaldifferent kinds of skin or nerve lesion (diseased places).Bacilli in a nerve can cause it to become thickened anddamaged. The muscles which it supplies become weak andthin. The patient loses the feeling in the part of the bodywhich it supplies. He may not be able to feel a piece ofcottonwool when it touches him (light touch) or a pin prick (pain).He may also not be able to tell the difference between a testtube of hot water and a test tube of cold water when theytouch his skin (the feeling of hot and cold). A part of thebody without any feeling is said to be anaesthetic. Wecannot say much more about the different kinds of leprosylesion here, but you should know the kind of leprosy inwhich you are likely to find bacilli in the skin or nose. Thelower a patient’s resistance the more likelv you are to$ndbacilli. Thus there are no bacilli to be found in the skin ofpatients with tuberculoid leprosy, but there are manymillions in the skin of those with lepromatous leprosy.The fight between man and Myco. lepraecan be looked atlikethis.METHODSFOR LEPROSY11.11 a Classifying leprosyAs we have seen, leprosy is caused by Mycobacteriumleprae, which is closely related to Mycobacteriumtuberculosis and is also acid fast when stained by theZiehl-Neelsen method. Myco. leprae is, however, notquite so acid fast as Mvco. tuberculosis. Smears forleprosy are thus stained for a longer time with carbolfuchsin and decolorized with weaker acid. BecauseMyco. leprae causes disease of the skin and nerves andoften the nose, it is most easily found in smears madefrom the skin or occasionally from the nose.When myco. leprae get into a patient a fight startsbetween the bacilli aild his body. What may happen inthis fight is shown in FIGURE 1 l-3. Many people fight thebacilli so well that they are never able to grow andmultiply and cause disease. These are the healthy peopleA. Resistance is so high that infection is never ableto start-the patient remains wellThe bacilli are unable to grow and multiply in thepatient, so no disease is caused and no bacilli can befound in the skin by the ordinary methods.B. Resistance high, but not so high as in A. so thatslow infection results-tuberculoid leprosy (TT)This kind of leprosy is usually mild, and it does notchange iuto the more serious kind. The most commonskin lesion is large, and has a raised edge. The skin of thelesion is paler than normal, thickened and anaesthetic. Alesion of this kind is called a tuberculoid plaque. Only oneor at the most two nerves may be thickened. Theythicken early on in the disease and on one side of thebody only. There are no bacilli in the skin scrapings.

HEALTHYvery highresistancePERSONThe differentkinds of leprosyHEALTHYPERSONthis is the easiest wayof classifying leprosymost cases of leprosy‘* start as indeterminateleprosybbacil~ot?%&foundthe fight between thepatient and the bacillistarts herebacillifoundnot usuallythis is a more completeway of classifyingleprosy9Borderline tuberculoid (BT)ITrue borderline(BB)Borderline lepromatous (BL)Leprosy is the result of a fight between leprosy bacilli and a healthy person.Many people have such a high resistance to leprosy [they are so good at fightingit) that the bacilli are never able to grow in them. Even though these peoplemay meet the leprosy bacilli, they stay healthy - Picture A. Other peoplehave less resrstance. lose the fight, and get leprosy. If they have no resistanceat all they get leprosy very badly - lepromatous leprosy - Picture 0. If theyhave some resistance they get it less seriously - borderline leprosy - Picture C.If they have a high resistance, they get a mild kind of leprosy - tuberculoidleprosy - Picture B.‘L LEPROMATOUSLEPROSYno resistancetFig. 11-3 The different kinds of leprosy

Classifying leprosy 1 11.1 laThese patients usually have anaesthetic patches but nobacilli. They are not infectious, and the disease sometimesdies out by itself, but this may take a long time andleave much disability because some nerves are damaged.C. A middle degree of resiktance-lborderlinerepros yPatients are often seen who are half-way between thetuberculoid and the lepromatous kind of the disease.These patients are said to have intermediate (middle) orborderline leprosy (also called dimorphous leprosy).This is a serious kind of the disease and may later changeinto the even more serious or lepromatous kind. Thiskind of leprosy is sometimes divided into three others,the middle one being the true borderline kind of leprosy-BB leprosy. Slightly less serious and more like tuberculoidleprosy is a kind called ‘borderline tuberculoid’ orBT leprosy. Slightly more serious and more like lepromatousleprosy is a kind called borderline lepromatousor BL leprosy. Bacilli can always be found in the skin ofpatients with BL leprosy. They can be found less often inBB leprosy and still less often in the milder BT leprosy.Any case of borderline leprosy may be infectious, especiallyBL leprosy.D. Very little resistance-lepromatous leprosy (LL)This is the most harmful kind of the disease; it remainsserious and does not change into the other less seriouskinds. Several nerves are usually involved equally oneach side of the body, but only late in the disease, andthere is generally not much anaesthesia until late on.Large numbers of bacilli may be seen in scrapings fromthe lesion, from the lobes of the ears or from the nose, oreven from healthy looking skin. These patients are openor infectious and do not get better unless they are treated.E. fndetarminate leprosyWhen a leprosy patient is examined, it is usually possibleto say that he has one of the above kinds of leprosy.However, there are a few patients, especially early on inthe disease, for whom this is not possible. These patientshave leprosy, but it is not the tuberculoid, nor the intermediatenor the lepromatous kind. They are thereforesaid to have indeterminate leprosy and are shown inPicture E in FIGURE 1 l-3. Indeterminate means undecidedand is quite different from intermediate whichmeans in the middle and which is the same as borderlineleprosy. Many patients with indeterminate leprosy getbetter by themselves, some of them stay indeterminate,and others change into the tuberculoid, borderline orlepromatous kind of the disease. Most of these patientsdo not have bacilli in their skin and are not infectious.F. Neural repros yA very few patients have leprosy of their nerves only,without any skin lesions, or any healed skin lesions.Perhaps this way of dividing or classifying cases ofleprosy looks too difficult. A simpler one is to divideall cases into indeterminate, lepromatous, and nonlepromatouskinds. In this way of classifying leprosy theborderline lepromatous (BL) leprosy is counted in withlepromatous leprosy, and all other borderline cases (BBand BT) are counted with the tuberculoid cases as beingnon-lepromatous. This easier way of classifying leprosycases is shown on the right hand side of FIGURE 1 l-4.The skin scraping in leprosy is useful for: ( 1) diagnosis;(2) finding if a case is infectious or not; (3) seeingif a case is active or inactive.( 1) DiagnosisLeprosy is diagnosed by finding one or more of thefollowing signs: (i) by finding skin lesions which areanaesthetic because the nerves to them have been destroyedby leprosy; (ii) by finding thickened and oftentender nerves which have become diseased with leprosy;(iii) by finding bacilli in the skin of the more seriouskinds of the disease. In tuberculoid leprosy anaestheticpatches will be found without bacilli in the skin. In lepromatousleprosy bacilli will be found in the skin, but therewill usually be little or no anaesthesia. A leprosy patienthas usually either anaesthesia or bacilli, but not oftenboth.(2) InfectiousnessPatients with many bacilli in their skin are likely to beinfectious or open and thus dangerous to other people.Those with few or no bacilli are probably not infectiousand are thus unlikely to spread the disease to otherpeople.(3) ActivitySkin scrapings also help us to know if a patient’s leprosyis active or inactive. By active we mean that his leprosyis getting worse or progressing. By inactive we mean thathis leprosy is healed. Leprosy is said to be active if anyof the lesions are raised above the rest of the skin, if theyare a red or coppery colour, or if they are increasing insize or number. The disease is active if an anaestheticpatch is getting larger, if the muscles supplied by adiseased nerve are getting weaker, or if a nerve is moretender than it should be when it is pressed. Leprosy isalso active when there are bacilli or the remains of bacilli(acid-fast debris) in scrapings from the skin or nose. Thisis because, when the dead remains of bacilli are seen,there may be living bacilli somewhere else that are notseen. Leprosy is inactive when none of these things arepresent.Leprosy patients should be treated with sulphones aslong as their leprosy stays active. This is seldom lessthan 2 years in tuberculoid cases and is at least 4 inlepromatous patients. Tuberculoid patients should betreated for at least 18 months after they have become

I ~z,:;~2,,,‘I I f.;-11 j Some Other Specimensinactive, and lepromatous patients for at least 5 years.Only when this long time of treatment is over canpatients be ‘released from control’ and allowed to goaway cur&11 .l 1 b The skin scrapingThe most important part of this method is the way inwhich the skin is scraped and smears made. The bestinstrument to use is a scalpel, which is a special kind ofknife used by surgeons. It is most important to choosethe right part of the skin from which to make the smears.You are most likely to tlnd bacilli in the edge ofan activelesion. As you have just read an active lesion is onewhich is spreading or raised or reddened, or in which theskin is thickened. By raised we mean that, as you moveyour linger over it, you can feel the edge of the lesion asbeing higher than the healthy skin. Also take a smearfrom the middle of the lesion, if it is flat (macular), or if itis raised equally all over, or if it is just a lump.METHODMAKING A SKIN SCRAPING FOR LEPROSY, FIGURE 11-4Take a clean slide without scratches. Choose the bestside on which to make your smears. Write the patient’sname in grease pencil on the other side where it will notbe washed off by the stain.1. Put several smears on a slide. There is room forabout six. Start at one end with a smear from the edgeof the lesion. If necessary take the next from the middleof the lesion (see above). Put next to it smears from thelobes of each ear. If you are going to do a nasal (nose)smear, do this next. Last of all. if there is room, takesmears from the skin of the buttocks or thighs. If thereare several lesions. start off with smears from two orthree of these and leave out the smears from the buttocks.If there are no skin lesions, take smears from thelobes of the ears. then the thighs, the arms, the back,and the buttocks.2. Take hold of some gauze with a pair of forceps. Dipit in spirit, and rub it firmly over the part of the body youare going to scrape.3. Take a sharp clean scalpel and dip it in spirit.4. Put the end of the scalpel into a flame for a secondor two. This could be the flame of a Bunsen burner, aspirit lamp, or a match. Don’t keep the scalpel in theflame for too long or it will get blunt-two or threeseconds is quite long enough. Never let the scalpel getred hot.5. Pick up the edge of the skin you are going to scrapein your fingers, and hold it firmly. This will help to stopit bleeding when you cut. Don’t let go until you havefinished making your smear.6. Make a shallow cut into the edge of the skin about5 mm long. This is as long as line A-B in this picture.Continue to pinch the skin together tightly, and only justcut into the top layer of the skin-you should just enterwhat is called the ‘papillary layer of the dermis’. Thereshould be no bleeding while you are scraping, and only avery little when you have stopped.7. Turn the scalpel sideways. Scrape the edges andbottom of the cut with the point of the scalpel. Start atone end of the cut, and scrape through to the other end.Then start at the beginning once more and scrape again.8. You will now have a little red pulp (soft red substance)on the end of your scalpel. Try to take the sameamount of pulp from each scraping you make, so thatyou are able to compare the number of bacilli in onescraping with those in another. Let go the skin and put apiece of dry cotton wool on the cut you have made.9. Smear this red pulp in a littie round area on yourclean labelled slide.10. Clean your scalpel in cotton wool and flame it asin steps 3 and 4 above. When your scalpel is clean andsterile again, make more smears from other parts of thebody, cleaning and sterilizing it between each smear.Put the smears on the same slide in this order: lesion(s),ears. nose (if necessary), and lastly the buttocks orthighs.If your smears are not already dry, let them dry andthen fii them by passing the slide quickly through theflame.If slides are not fixed at once, keep them in a boxaway from dust and insects. If they have to be sent toanother laboratory, fii each slide and wrap it separatelyin a piece of paper before sending it off.Stain your slide by the Ziehl-Neelsen method. butleave the carbol fuchsin on for 10 minutes with the hotmethod, and 30 minutes with the cold method. Decolorizewith 1% acid-alcohol with both methods.Search the slide for AAFB starting with the smearsthat came from the lesions. Myco. leprae looks likeMyco. tuberculosis, except that it is more often beadedand may be found in groups or clumps (globi).ALWAYS STERILIZE YOUR SCALPEL BETWEENONE PATIENT AND ANOTHER. IF YOU DON7 YOUMAY GIVE SOMEONE LEPROSY WHO HAS NOTALREADY GOT IT.11 .11 c Nasal smearsBacilli can usually be found in the nose of patients withlepromatous leprosy. But bacilli can be found in the skinof these patients just as easily. A nasal smear may also beuncomfortable for the patient, but if it is done carefully itshould not be painful. When AAFB from the nose are inglobi we can be sure the patient has leprosy. But, whenthey are not in globi they may be the harmless mycobacteriadescribed in Section 11.3. For these reasonsnasal smears are not done as often as skin smears, andyou should not do them unless you are specially asked to.The main reason for doing them is that bacilli may stayin the nose after they have gone from the skin. When apatient is relapsing (getting worse again after starting toget better) bacilli may also come back in the nose beforethey come back to the skin.

‘,y,d+“,> &^-”,.,-:>. I -_’ ‘.6-WHERE To MAKE IHE sm lEAR/ hand -ear-leprosy lesionleprosy lesion1 \I b-healthy skinflameskin until you haveyour smeara match,Bunsen burnera paraffin_pressure stove,or a spirittlamp canbe usediif&Fleprosy lesionorcepsthe finshedturn the scalpel ! .sideways like thisedge of first1lesion10slide”

f 1 1 Some Other SpecimensNasal smears can be made in various ways. A woodenapplicator stick round the end of which is a little cottonwool can be used. The best instrument is probably aspud. This is a small spade with which the nasal mucosacan be scraped. A useful spud can easily be made from apaper clip or a bicycle spoke.METHODMAKING A NASAL SMEAR. FIGURE 11-5Ask the patient to blow his nose to blow away anymucus. If he has no handkerchief, ask him to blow hisnose in toilet paper or in his fingers.1. Take a bicycle spoke or a strong paper clip. In thisfigure a paper clip is used.2. Straighten out the paper clip.3. Hit the end of the paper clip on a stone with ahammer until its end is flat.4. The edge of the paper clip should be sharp, but ncittoo sharp. This is now your spud.5. You will want a special instrument called a nasal(nose) speculum to put into the patient’s nose.6. You will need a good light, so move the patient’shead until light falls on his nasal septum. This usuallymeans bending his head backwards.7. Pinch the speculum together.8. Put the end of the speculum into the patient’s noseand leave it there. The speculum will open and its endswill hold his nose so that you can easily see inside.9. Meanwhile flame your spud and leave it to cool.10. Put the cool spud into the patient’s nose andgently scrape an area of thenasalseptum. The nasal septumis the middle part of the nose which divides oneside from the other. When there are leprosy bacilli in thenose the mucosa is usually reddened and thickened, andthere may be small yellow swellings called nodules. Tryto dig out a small part of one of these nodules. Theycontain millions of bacilli. If there is an ulcer (open soreplace) on the septum. scrape this.Don’t scrape the skin of the lower part of the inside ofthe nose. This part of the nose, where you can easily putyour finger, is called the vestibule (entrance). You willnot usually find mycobacteria here,11. Scrape the mucosa once or twice until you have alittle soft red pulp on the end of your spud. Don’t scrapeso hard that the mucosa bleeds more than a little. Bleedingspoils the chances of your finding bacilli.12. Spread this soft red pulp in a small circular areaon a slide. Nasal smears are usually put on the sameslide as smears from the skin. Stain nasal smears forMyco. leprae in the same way as you would a skinsmear.11.1 Id Examining and reporting on smears forM yco. tepraeSearch the smears you have made for bright red bacilli inthe same way that you would search sputum smears forMyco. tuberczdosis (Section 11.1). Look at not less thm100 fields, unless the patient is lepromatous, when therewill be millions of bacilli, and one field will be enouigh.You may find a few separate bacilli, or there may; bemany of them close together. Many bacilli close togetherinside a cell are called globi and are found in lepromatousleprosy. If you find a positive slide, keep itif possible so that it can be checked. After it ‘hasbeen checked, boil it in dichromate (see Section 3.12)or break it so that it cannot be used again. Thiswill help to prevent false positive reports-see Section11.2.Leprosy smears can be reported in several ways. Weshall explain two of them, and you must use whicheveryou are asked to.WHO’s method of reportingNegativeOne plus (+)Two plus (+ +)Three plus (+ + +)Four plus (+ + + +)Ridley’s method of reportingNo bacilli in 100 fieldsOne or less than one bacillusin each fieldBacilli found in all fieldsMany bacilli found in allfieldsMany bacilli and many globiNegativeNo bacilli in 100 fieldsOne plus (+) l-10 bacilli on average in100 fieldsTwo plus (+ +) I- 10 bacilli on average in 10fieldsThree plus (+ + +) l-10 bacilli on average inone fieldFour plus (+ + + +) 10-100 bacilli on average inone fieldFive plus (+++++) 100-1000 bacilli on averagein one fieldsix plus (++++++) Many clumps or globiAs we have seen above, a patient’s leprosy is stillactive if the debris or remains of bacilli can still be foundin his skin or nose. If, therefore, in patients under treatment,you see acid-fast debris which looks like theremains of bacilli, report ‘acid-fast debris present’. Makesure, however, that this is not just a deposit of carbolfuchsin on a badly made slide.The bacteriological indexAs well as reporting on each smear in one of the aboveways, you may be asked to work out what is called thebacteriological index. It is a measure of the total numberof live and dead bacilli in a patient. This is very easy andis only a way of averaging out the ‘number of plusses’ ina patient’s smears. It is a useful way of showing howmany bacilli a patient has. Examine the smears from each

$i&i: :: -&ll _ ~*”ii’1~11’ 1 Some O&r Specimenspart of the patient’s body in one of the ways describedabove. Add up the number of plusses and divide by thenumber of smears you took. For example, you might get2+ from the edge of the lesion, 3+ from the middle of thelesion, 4+ from the right ear, 2+ from the left ear, l+from the nose and none from the buttocks. This makes atotal of 12 plusses from 6 smears. The bacteriologicalindexisthus 12+6=2.The morphologicalindexAs we saw earlier on, a fight goes on inside a leprosypatient between his body and the bacilli. Bacilli whichare growing and multiplying and so winning this fightstain as uniform dark red rods. By uniform we meau thata bacillus looks solid and stains in the same way all over.By dark red we mean that the bacilli are a dark redcolour and are not pale. Bacilli which stain as uniformdark red rods are often called solids. But, if the bodyw’lns the fight and the bacilli start to be killed, they nolonger stain in the same solid uniform way. As it dies, arod-shaped leprosy bacillus starts to stain irregularly (inlumps) and then breaks into granules, as shown inPicture A, FIGURE 11-6. Dying bacilli may also stain apaler pink instead of a dark red. Pale oi granular bacillilike this are often called non-solids. Only solid dark anduniformly staining bacilli are alive. Pale or irregularlystained granular non-solids are dead. The body takes along time to get rid of dead leprosy bacilli. This is whynon-solids and acid-fast debris can be found in skinsmears long after treatment has started and the patient isgetting better.The number of solid rod-shaped live bacilli in everyhundred of all kinds (alive and dead) is called the morphologicalindex. It measures the percentage of livebacilli in a smear. The higher the morphological index(the nearer 1009/o), the more live bacilli there are, and themore infectious a patient is. A lower morphologicalindex shows that there are fewer live bacilli and that treatmentis making the patient better.Even in the case of lepromatous leprosy not all thebacilli are alive, and even in an active untreated case themorphological index may only be 30% or 40%.The morphological index is a good way of seeing howthe fight between the patient and his bacilli is going. Likethe bacteriological index, which is a measure of all bacilliin a smear, alive and dead, it tells us if the patient iswinning or if the bacilli are winning. The morphologicalindex is the better way of telling how the earlier stages ofthe fight are going. Let us say that a lepromatous patientstarts treatment with sulphones. Most of his bacilli arekilled in a few months; so they stain irregularly. Themorphological index, which might have been 30% beforetreatment started, falls to 5% or even 0%. The bodytakes a long time to remove dead leprosy bacilli, so thebacteriological index, which was, say, 5 before treatmentstarted, may only fall to 4. This is because the bacteriologicalindex measures all bacilli, alive and dead. Themorphological index, which falls so soon after suc-cessful treatment has begun, is thus a more sensitiveindex of response to treatment than the bacteriologicalindex.Even though the. sulphones can help the body to killthe bacilli and the morphological index falls to 5% oreven 096, the few that may remain hidden in the liver andthe nerves can grow and multiply if the sulphones arestopped. This is why it is so important for a patient to goon taking sulphones for years alter his skin smears arenegative, so as to make quite sure that there are no livebacilli left in his body.Leprosy bacilli may become resistant to the drugsused to treat the patient. Sulphones, which used to killthe bacilli, may no longer do so, and the bacilli are saidto have become resistant. When this happens, the morphologicalindex, which may have been 0% soon after thetreatment started, rises again. The morphological indexis thus a good way of telling if a patient has becomeresistant to his drugs. He must of course have beentaking his drugs regularly, and this is why it is useful tobe able to test his urine for sulphones as described inSection 8.1Oa.METHODTHE MORPHOLOGICAL INDEX. FIGURE 11-6Choose e well-stained part of the film.Find 100 separate organisms and count how many ofthem are solid, rod-shaped, and deeply and uniformly3. A C. A globus D. A clumo of bacilliinside a cellA.The death of aslive leprosy bacillus-s0lid Plates 105 - 107 F. ‘non-solids’dead 2’ deadgone,‘CFig. 1 l-6 The morphological indexQ granulesLodgLstained like those in Picture E. These are the solids.Don’t count any organisms in globi or clumps, like thosein Pictures C and D. If an organism is pale or is veryshort and granular, it is a non-solid. If you find a longerthan average solid bacillus with a short gap in the middle,it is probably about to divide into two. Count it astwo solids. If you see a row of two or three granulestogether, which are the remains of one bacillus, callthem one non-solid.

icycle spokeflatten out 1this is the end ofthe paper clipfrom the sidethis is a nasal speculum,-put the nasal speculuminto his nose\THIS IS A VIEW LOOKINGINTO THE NOSE77h0I I> I I’ 4gl I :I1 ‘i. J ’Ii2’1 ’ 11flameyourspudfbend the patient’shead backwards\ /thickened patchsoft red pulp’I the mucosa isshown whitehere, in thelabel the slidebefore you makethe filmsmfrom from fromlesion ear noseflame your film and stain itby Ziehl-Neelsen’s methodFig. 1 l-5A nasal smear for leprosy

-.,‘&A, ,1 ’ .C’. ‘--Lymph node puncture for trypanosomes 1 11 .12METHODS FOR SOME OTHER DISEASES11.12 Lymph node puncture for trypanosomesThe lymph nodes (lymph glands) are about the size andshape of a bean. There are some in the neck, some underthe arm, and some in the groin. The groins are the foldsbetween the abdomen or belly and the legs. There aremany more lymph nodes deep inside the body. Lymphnodes filter lymph (fluid from the tissues) and are one ofthe places where lymphocytes come from.When a patient is infected with a species of trypanosomecalled Trypanosoma gambiense, the lymph nodes atthe back of his neck often become swollen (large). Trypanosomescan otten be found in these swollen glands bypuncturing them. (making a hole in them) with a needleand sucking out some blood and lymph with a syringe.Moving trypanosomes can be seen, just as in blood (seeSection 7.36) or CSF (see Section 9.14).METHODPUNCTURING LYMPH NODES TO FIND TRVPANDSOMESCarefully explain to the patient what you are going todo.Fetch slides, coverslips. a sequestrene bottle, swabs,spirit, and a syringe with a sharp sterile needle.Sit the patient down with his back to you. Feel for thebiggest node at the back of his neck. Swab the skin overit with spirit.Take the syringe in your right hand and pinch the nodebetween your left finger and thumb.Put the needle into it and try to suck out some fluid.Put the first drop of any fluid you get on to a slide. Putthe rest of the blood into the bottle, if there is any. Put acoverslip on the slide.Swab the skin again and put some adhesive plasteron the wound.Look for moving trypanosomes all over the film, just asyou would in blood (see Figure 8-l 4). If you can only get avery small drop of fluid, add a smalldrop of saline, or it willdry up.If you cannot find trypanosomes on the slide, and youhaveenough bloodinthebottle,spinitandlookatthespun~deposit.When you try to suck fluid from the node you maythink you are not getting any. But there will probably be asmall drop in the needle. Blow this on to a slide and lookat it. It may well be enough to let you find trypanosomes.11 .13 The rectal snip for Schistosoma mansoniThe trematode worm called Schistosoma mansoni livesin the veins of the large intestine (large gut). The femaleworm lays eggs in the veins which slowly push their waythrough to the inside surface of the gut. These ova arethen passed out of the body in the stools where they canbe found either in a saline smear (see Section 10.2) or bythe concentration test described in Section 10.3. Thisconcentration test is probably the best way to find theova of S. mansoni. But some people like to diagnose S.mansoni infections by looking at a small piece of rectalmucosa called a rectal snip. They put a special short,wide metal tube called a proctoscope (or a longer onecalled a sigmoidoscope) into the patient’s anus (the bottomend of the large intestine). They look through thisand take out a small piece of the mucosa of the largeintestine with special forceps. This piece of mucosa isthen squashed between two slides and looked at for thelarge lateral (side) spined ova of S. mansoni. Most of thismethod is for doctors.METHODTHE RECTAL SNIP FOR S. MANSONI-FOR DOCTORS ONLYPass a sigmoidoscope or a proctoscope. Look for apiece of mucosa that looks abnormal. Remove a smallpiece of the mucosa with a pair of forceps. If you cannotfind a piece of abnormal looking mucosa, take a piece ofnormal mucosa. Don’t take too large a piece or the gutwill bleed too much. Put it on a slide. Add a drop ofsaline and put another slide on top of it. Squeeze thetwo slides together and look at the piece of mucosathrough a low power objective. Look for schistosomeova. Either the ova of S. haematobium, or the ova of S.mansoni may be found, or possibly even both. These ovaare shown in Figure 10-8.11 .14 The skin snip for Onchocerca volvulusOnchocerca volvulus is a worm which lives in the skinand causes a disease called onchocerciasis. The wormmakes lumps on the skin called onchocercal nodules, andit also makes patients blind. The female worm gives birthto many young worms called microfilariae. The microfilariaeof most nematode worms which live inside the‘ body tissue are found in the blood. These blood microfilariaeare described in Section 7.37. The microfilariae ofq’ 0. volvulus are different and are found in the skin. We cantherefore diagnose onchocerciasis by cutting a very smallpiece of the patient’s skin and looking for microfilariae init with a microscope. If we watch we can see live microfilariaecome out of the piece of skin and move about in adrop of saline under a coverslip. This is called doing askin snip for onchocerciasis. Use a sharp scalpel and asharp needle held in a loop-holder. If you do many skinsnips keep a special loop-holder and needle for thesealone. A sewing needle can be used, so can one of theneedles that fit on to a syringe. But the best k ind ofneedle to use is called a ‘Hagedorn’ needle and is used bysurgeons for doing operations. Ask someone in theoperating theatre of your hospital if they can give youa small Hagedorn needle. Put this into the end of a loopholder;you may have to break the end of the needle toget it in. This needle is for lifting up the skin while youcut off a very small piece with a scalpel.

Eneedle\hold the needle flatclose to the skinF foldof-lift upthe needlebottleof salineGskin snipthe dotted lineshows the cut&a--r-rslidepiece of skinTHIS IS A LOW POWERMICROSCOPIC VIEWmicrofilaria coming out 1of the skininered cells which havecome from the tissueL mrcrofilaria of Onchocerca voIvuIusFig. 1 l-7The skin snip for onchocerciasis

‘ h-7 1‘, ,,.->$ ,’ .: 1,:. + _,:’ ‘.‘;Micro6lariae are more likely to be found in skin snipstaken from special parts of the body. In men we usuallytake skin snips from the skin over the iliac crest. Theiliac crest is the top of the bone called the ilium which ispart of the pelvis. Picture A, FIGURE 1 l-7, shows a skinsnip being taken from the skin over the iliac crest of aman. The middle part of Picture A has been drawnmuch larger in Picture C. Here, the needle is holding upthe skin and the scalpel is just going to cut off the skinsnip.In women it is usually easier to take a skin snip fromthe skin of the back of the shoulder. This is being done inPicture B.If you only take a small piece of skin, there is no needto use a local anaesthetic. This is a drug which is injectedinto the place to be cut to stop the patient feeling pain.scrapings of skinon a slidearmMETHOD_ SKIN SNIPS FOR ONCHOCERCA VOLVWUS. FIGURE 11-7If possible send the patient into the sun for half anhour and let the sun shine on the skin where the snipis to be taken. This will make it easier to find the microfilariae.Swab the skin with spirit Let the spirit dry.Quickly flame the needle and let it get cool. Don‘t letit get red hot, or it will get blunt.Push the needle into the skin where you want to takea snip. Hold it flat and only just push the point in. This isshown in Picture E.Lift up the needle as shown in Picture F. This willraise a fold of skin.Cut off the skin just under the needle with a sterilescalpel or a sterile razor blade. This is shown in PictureG. The dotted line shows where the scalpel has gone.The skin snip will stick to the needle.Put the skin snip on a slide. Put a drop of saline on it.Cover it with a coverslip. Ring the coverslip with paraffinwax and Vaseline as described in Section 7.23. Thiswill stop the saline drying.add a drop of 20% potassiumsodium hydroxideput a coverslip on themixtureorLook at the skin snip with your, microscope. Use a lowpower objective. You will see small worms come out ofthe piece of skin and start moving about in the saline.These worms are the microfilariae of 0. volvuZus andhave been drawn in Picture r). Some worms will comeout of the snip very soon. If you don’t see any worms atfirst, look again 20 minutes later. Don’t call any specimennegative until you have looked at it after it hasbeen taken for 20 minutes. Count the microfilariae andreport the number you see.11.15 Skin scrapings for fungiMICROSCOP’Cbranching mycelial:&filament LosAngthe borders ofcells/VIEWheat for a moment untilthe liquid just boilsWhen food such as bread is left in a damp warm placefor a few days it usually becomes covered with manyvery short thin hairs. These may be white or colouredand are often green. They are the hairs or mycelia ofFig. 1 l-8 Skin scrapings for fungi

;L, -,(/^ ,# e

This chapter makes extensive use of material and drawingskindly supplied by Dr Jean Holland, formerly of theUganda Blood Transfusion Service.12.1 Blood groups and agglutination (FIGURE 12- 1)In the other chapters of this book you have read aboutthe methods we use to find out what disease a patient has(to diagnose his disease). For example, you will haveread about measuring the haemoglobin and looking forhookworm ova in the stools. This is important because, ifyou can find out why a patient is ill, he can be given theright treatment and often be cured. Blood transfusion isnot done to diagnose a patient but to treat him. Bloodtransfusion means taking blood from a healthy personand giving it to a patient who has not got enough blood.The patient to whom the blood is given is called a recipient(recipient means receiver). The healthy person whogives blood is called a blood donor (donor means giver).A blood donor soon makes some more blood to replace(make up for) the blood he has given and a donor cansafely give blood every 6 months.Blood transfusion saves many lives, but it can be dangerousboth to the donor and the recipient, unless it isvery carefully done by someone who has been trainedin blood transfusion work. This chapter explains theeasier parts of blood transfusion, but the only wayto really learn how to give a safe transfusion is to betrained in a good laboratory by someone who is experienced.We cannot take blood from any donor and give to anyrecipient. This is because there are many different kindsof blood, even though every healthy person’s blood looksthe same. We call these different kinds of blood the bloodgroups.Some blood groups can be mixed together and are saidto be compatible but other blood groups cannot be mixedand are said to be incompatible. When a recipient needsto be given a blood transfusion, he must be given bloodwhich will mix with his own blood. If a recipient is giventhe wrong kind of blood he may die. There are specialmethods for finding out the group to which bloodbelongs. We have first to find the recipient’s blood group,and then 6nd some blood from the right kind of donorwhich will mix with his blood. There are many bloodgroups. We shall only describe the most important ones.There are.the ABO blood groups and the Rhesus or Rhblood group.There are four important kinds of ABO group: A, B,0, and AB. There are two important kinds of Rhesusgroup: Rhesus positive and Rhesus negative. Sometimes,instead of saying Rhesus positive, we call the blood D(‘big D’) positive.A person with any kind of ABO group can be eitherRhesus positive or Rhesus negative. People can thereforehave these kinds of blood:Percentage of peopleof each blood groupABO group Rhesus group (Zambia)Group A Rhesus positive 25.33Group A Rhesus negative 0.67Group B Rhesus positive 21.43Group B Rhesus negative o-57Group AB Rhesus positive l-95Group AB Rhesus negative 0.05Group 0 Rhesus positive 48.69Group 0 Rhesus negative 1.31The blood groups are not all equally common. In thelist above you will see the number of each blood group inevery hundred (the percentage) that we find here inZambia. In other parts of the world the percentage ofpeople in each group will be slightly different. You willsee that group 0 is the most common group, and thatthere are many more Rhesus positive people than thereare Rhesus negative people.A person’s blood group is the same all his lge. Theblood groups are like the colour of a man’s eyes or hisskin: we are given them by our parents. Nobody everchanges his blood group.You have read that blood contains red blood cellslying in a liquid called plasma. People of different bloodgroups have different red cells and different plasma. Thered cells of each blood group are different because theyhave different ‘places’ of a special shape (called determinantgroups). Group A red cells have A places. Group Bred cells have B places. Group AB red cells have both A

12 1 Blood Transfusionand B places. Group 0 red cells have neither A nor Bplaces.The plasma of people with dil%erent blood groups isdifferent because it contains special proteins calledat&Iii. These antibodies are long proteins with a‘hook’ (calld a valency site) at each end. The hooks onanti-A antibody hook on to A places on the red cells. Thehooks on mti-B antibodies hook on to B places on redcells. There is no common anti-0 antibody. If you lookat a drop of blood through a microscope, you can see thered cells, but antibodies are so small that they cannot beSeCSA.Because each antibody has two hooks which can hookon to two red cells of the same group, antibodies canmake red cells stick together in lumps or agglutinates.We can watch this aggIutInatIon or clumping on a slideor a tile as in FIGURE 12- 1. Picture L shows a tube withanti-A serum in it. Picture M shows a tube containingsome group A red cells. Picture N shows a drop of anti-A antibody and a drop of group A red cells being mixedon a tile. A minute or two alter they have been mixed thecells will be seen to ccme together in lumps or agglutinates.These agglutinates are shown in the right hand sideof the tile in Picture N. In the middle part of this figureis a diagram to help you learn what happens. Picture 0 isan antibody: it is anti-A. You will see that it is long andhas the same kind of hooks (valency sites) at each end.Picture P is a group A red cell with special group Aplaces (determinant groups) which fit the hooks on theanti-A antibody. The anti-A antibody is really very muchsmaller than the red cell and has been drawn much toobig in these pictures. When anti-A antibody and group Ared cells are mixed, they hold together to form an agglutinate,as shown in Picture Q. Pictures R and S showanti-B antibody and group B red cells. Anti-A only agglutinatesgroup A and group AB red cells. Anti-B onlyagglutinates group B and group AB red cells. We saythat these sera are specific for these blood groups.Healthy people never have in their plasma any antibodyagainst the special places on their own red cells, butthey always have antibodies against the special places ofthe other ABO blood groups. This means that group Apeople who have red cells with special A places haveanti-B antibody in their plasma. Group B people whohave red cells with special B places have anti-A antibodyin their plasma. Group 0 people have red cells withouteither of these special places; so they have both anti-Aantibody and anti-B antibody in their plasma. Group ABpeople have both A and B special places on their redcells; so they can have neither anti-A nor anti-B antibodiesin their plasma.In this example the red cells are acting as antigens.You may have heard of antigens before. When bacteriaenter the body, they act as antigens, and the body makesantibodies against them. These antibodies ‘attack’ (joinon to) the bacteria and help the body to fight them. Mostantibodies are only formed after an antigen has got intothe body. Anti-A and anti-B antibodies are different. Weare born with them.12.2 TransfusionIn a blood transfusion a donor’s red cells must never beallowed to agglutinate inside a patient. The donor’s redcells must therefore be compatible with the patient’sserum. If they are incompatible, they may agglutinateinside the patient who will have a traasfiwion reaction.He may shiver, have a fever, become jaundiced, and hemay have pain in his back. His kidneys may stop workingbecause. they are blocked by agglutinated red cells.When a patient’s kidneys stop working he passes nourine and is said to suffer from anuria (no urine). Becausepatients often die from transfusion reactions, wehave to take great care to prevent agglutination of theirblood.Because group AB patients have no anti-A or anti-Bantibodies in their serum, they can be given blood of anygroup. They are called universal recipients (receiversfrom everyone). Because group u red cells have no specialA and B places on them, they can be given to anyone.Group 0 donors are therefore called universaldonors (givers to everyone). Group A people must thereforebe given group A or group 0 blood. Group B peoplemust be given group B or group 0 blood. Group 0people MUST ONLY be given group 0 blood. GroupAB people can be given blood of any group. Even thoughwe can give group 0 or universal blood, it is alwaysbetter, if possible, to give blood of the right(homologous) group. This means giving only group Ablood to group A patients, and only group B blood togroup B patients, etc. But universal donor blood is oftenuseful. Many hospitals always keep a bottle of group 0blood ready in a special refrigerator. If a patient is bleedingto death rapidly, he can be given this group 0 universaldonor blood without wasting time finding out whathis own blood group is. Later he can be given blood ofhis own group. Always take a specimen of the recipient’sblood into a dry tube before giving him universal donorblood in this way. It is often useful to be able to test thedonor’s blood before it has been mixed with universaldonor blood.12.3 AntiseraAntisera are used for grouping blood. Anti-A antibody iscalled anti-A antiserum or ‘anti-A’. Anti-B antibody iscalled anti-B antiserum or ‘anti-B’.The anti-A antiserum we use for blood groupingcomes from the serum of group B donors. Anti-B antiserumcomes from the serum of group A donors. Thesedonors are specially chosen so as to have strong antisera.Large laboratories choose their own special donors andmake their own antisera, but small laboratories have tobuy their antisera. Liquid antisera can be bought insmall bottles which often have small pipettes in theirstoppers. Anti-A is often coloured blue or green, andanti-B is often coloured yellow. Sometimes the sera aresold dry and are sealed (closed) into small glass tubes.These tubes must be opened and water added to them

this is a tube of \anti-A serum, it \ #etcontains anti-Aantibodydrnn nf rerl cell -suspension has beenolaced on the tileY --.-r-. .----.- -a drop of anti-Aserum is beingadded to theshort wide strong pipettea minute or two later, afterthe tile has been gentlyrocked (moved), the cells willstick together and look likethisthis is a washedtina Itionthis is ananti-A -rantibody0group A hookor valency sitePAL this is the patient’s name -\ this is a plastic tile, a white builder’stile can be used, so can a piece of plasticsheet, or a microscope slideantibodyTHIS IS A DIAGRAM OF WHAT HAPPENSQ be*T?jPlate12the hooks (valency sites)on an anti-B antibody and thespecial places (determinantgroups) on the group B redcells are different from thoseon anti-A and group A red cellsgroup B hookor valency sitegroup Bantibodygroup A determinantgrowgrowgroup Bred cellAn Eldon cardthis means that these cards are good this is the control square : if you seeenough for testing the Rhesus groupagglutination here it means that theof patients, but should not be used results in the order squares may be wrong :for donors . use lrqurd anh-D serum try the test again using a CONCENTRATEDfor rhesus testing‘donors,/rcard suspension 1 of washed red cells on anothera drop of antiserum has ~-been dried on each of these square S/thissquare forfinding the rhesus grouppatient’sname HIfollow the instructions very carefully !!!Fig. 12- 1 Agglutination

.,“” _; ; i ; _,I,>&?!, I--il.12 1 Blood Transfusion(distilled water is the best, and you must not use saline).The water mixes with the powder in the tube and makesliquid antiserum ready to use. Read the instructions thatcome with these tubes carefully.Most of the chemicals in the main list will keep goodfor ever. Whenever we want to use them we can be surethat they will work. But anti-A serum and anti-B serumare not like this. Antisera very easily go bad becausemicro-organisms grow in them. They may then stopworking. ALWAYS KEEP YOUR ANTISERA IN AREFRIGERATOR. If you have a store of antisera thatyou are keeping for some time, keep it frozen solid in thecoldest part of the refrigerator (the ‘freezer’; see FIGURE12- 11). Keep the antisera that you are using every day inthe main part of the refrigerator. It will stay liquid.But if antisera so easily stop working, how do wemake sure that the antisera we use are still working? Wedo this by using what are called controls. We test ouranti-A with some red cells that we know are group A andsome that we know are not group A. If we get goodagglutination with group A cells, but not with 0 or Bcells, we know our anti-A must be working. If our anti-Adoes not agglutinate A cells, we know it is not working.We test our anti-B in the same way with group B cells.Good laboratory workers do a control every time theygroup someone’s blood.But where can we find red blood cells that we can bequite sure are group A or group B? The best thing to do isto make sure that you know your own blood group and theblood groups of some of the other people working in thelaboratory or the hospitd, especially those people who arelikely to stay in the hospital some time. Among them youwill find some who are group A and some who are groupB. Put a drop of blood from the ear of the person withthe cells you want into a tube of saline. Centrifuge itto wash the cells, and re-suspend the deposit in justenough saline to make a good suspension for testing.But how do we make sure that the people whose bloodwe are using really are group A or group B? If possible,send their blood to a big laboratory where it can betested, and the answer can be sent back to you. If this. isimpossible, test the blood groups of the hospital staffwith strong new antiserum. If you group several peopleand some come strongly ‘A’ and others strongly ‘B’, youcan be sure the antisera are working well.You can keep these A and B cells for doing controls inthe refrigerator. It is best to keep them in bijou bottles of‘acid-citrate-dextrose’ solution that is used to keep blood(see Section 12.9). But, unless they are sterile (seeSection l-20), these samples will not keep more than aday or two, even in the refrigerator. Blood cells that areold and infected (see Section 1.15) may give the wrongblood group. Where you can, therefore, always use freshcontrol A and B cells.12.4 Washing red cellsYou might think that the easiest way to group bloodwould be to put two separate drops of the patient’s bloodstraight on to a tile, and to add anti-A serum to one dropand anti-B serum to the other drop. We can do this, but itis a bad way to group blood because we sometimes getthe wrong results. It is very important to ‘wash’ red cellsbefore they are grouped. By washing red cells we meanwashing away all the serum that came with the cells. Thisserum may cause the wrong results in blood grouping.Washing red cells is very easy. Add two or three dropsof blood (not more) to a tube full of saline. We use saline,not water, because the cells would haemolyse in water.Then centrifuge the tube of blood and saline. There willthen be some nearly clear supernatant saline and a smalldeposit of washed red cells. Take off all the supernatantwith a pipette. The serum you want to remove is in thissaline. Then add just enough clean saline to make theright strength of suspension for blood grouping. Thissuspension should be about 5% cells and 95% saline.Add about twenty times as much saline as red cells. It isnot easy to measure, and the strength of the suspension isnot important, but don’t make the suspension too weak.You now have the ‘washed cell suspension’ which is usedfor blood grouping and cross-matching (see Section 12.6for the meaning of ‘cross-matching’).12.5 Blood groupingIt is quite easy to find out the group of someone’s blood.All we need do is to mix on a tile one drop of ourpatient’s red cells with anti-A antiserum and anotherdrop of his red cells with anti-B antiserum. These are thethings that may happen:The patient’sAnti-A Anti-B blood group is:Agglutination Nothing Group ANothing Agglutination Group BAgglutination Agglutination Group ABNothing Nothing Group 0If nothing happens when we mix our patient’s bloodwith either anti-A or anti-B, his group is group 0. If hisblood agglutinates with both anti-A and anti-B, his groupis group AB. If it agglutinates with anti-A, he is group A.If it agglutinates with anti-B, he is group B.You will now be able to understand how to group apatient’s blood. You will need a good strong Pasteurpipette, a cup of clean saline and a cup into which to putthe waste saline. Read how to wash your Pasteur pipettein Section 3.9. It is also useful to have a wash bottle filledwith saline (see Section 3.40). The cups for saline areshown in FIGURE 3-6 and in FIGURE 3- 11.The quickest and easiest way of grouping blood is touse a tile. The most accurate way is to use a tube.METHODSGROUPING BLOOD ON A TILE. FIGURE 12-21. Fill a Kahn tube or a centrifuge tube with saline.Label it with the patient’s name. In Figure 12-2 thepatient is called Phiri.

.bthis is awash-IvlttlPof salinewith..--..-a few drops of bloodare being added to thtest tube, these can befrom a clotted or anunclotted specimenshort wide strong pipettethis is a test -tube being filledwith saline 0tube is labelledpatient’s nameathe suspension ofred.cells in salineIS being centrifugedr--the supernatant salinehas been removed leavingonly the deposit of red\ cellsa few drops ofclean saline arebeing added tomake about a 5%suspension the suspension ishainn r1wlth-4 intnthe pipettethere is a demsitof red cells and aclear supernatant I sb I L/washed cellsuspensionclear , or almostclearsupernatantsalinedeoositMAKE SURE YOU DO CONTROLStwo separate drops of thewashed red cell suspensionare being put on the tilethis is the plastic tilea drop of anti-B serumis being added to the \right hand drop, a dropof anti-A serum has alreadybeen added to the lefthand drop9V these ai paintedwith yellow paint0 (///.lj% oZthe ti,eafter adrlinnthnof plastic sheet , or a microscope slide the patient’s name;;;;vr$,L;;y 10 A MINUTE OR TWO LATER THIS IS WHAT YOU MIGHT SEEbefore reading theansweruniversalblood/G 7OUP A GROUP ABrecipientanti-A has been\added -anti-B h\as been added anti-A has been added -anti-B has beei addedFig. 12-2Blood grouping

12 1 Blood Transfusion2. Add two or three drops of blood. This can befrom the ear (see Section 4.7). from a sequestrenatedspecimen of blood, 6r from a clotted specimen. If youuse a clotted blood specimen. break up the clot withyour pipette and take some of the liquid blood from thebottom of the bottle. Try not to take up any clot intoyour pipette.3. Centrifuge the suspension of blood and saline.4. After centrifuging there will be a little deposit ofcells at the bottom of the tube.5. Take away the supematant saline with a Pasteurpipette.times as much saline as thereIi ‘make a 5% solution. Don’tmake the suspension too weak. This is your *washedcell suspension’.7.Take up some of the red cell suspension intoyour pipette.8. PLY two separate drops of the r;uspension ondifferent parts of a tile. If you have a plastic tile withdepressions (holes) in it (ML 49) use this. You canalso use a slide, as shown in ,picture 18, Figure 12-3.Using a clean pipette, put two separate drops ofgroup A red cells below’ the drops of blood to betested. Wash your pipette again, and put two separatedrops of group B blood below the group A redcells. These are your controls. They have not beendrawn in Figure 12.2.9. Add one drop of anti-A to each of the three ieft-handdrops of cell suspension, and one drop of anti-Bto each of the right-hand drops of suspension. If the seraare coloured, ariti-A is blue or green and anti-B isyellow. Make sure you do not muddle up the pipettesand the bottles. In Picture 9. Figure 12-2, the cap ofthe anti-A bottle with the pipette in it and the labelon the bottle are both painted with blye paint. Theanti-B pipette and the anti-6 bottle are labelled inyellow. This helps to stop them being mixed up.10. Gently rock (move) the tile from side to side. Ifthe blood is group 0 nothing will happen. If theblood is group A, B. or AB. it will agglritinate in theway shown in Picture 10. If the blood is going toagglutinate it will agglutinate soo+in about 2 minutes.Don’t let the blood dry up. and don’t leave itlong= than 5 minutes before reading the bloodgroup.Always check that the group A red cells and the groupB red cells give the right results. If they do not, youcannot tell the group of the patient.AGO TUBE GROUPINGPrepare a washed 5% suspension of red cells as insteps 1 to 5 above. Add two drops of washed redcell suspension to each of two carefully labelledtubes. To one add two drops of anti-A. To the otheradd two drops of anti-B. Leave them on the benchfor 2 hours. At the end of this time look for agglutinationunder the microscope.REMEMBER WHAT YOU READ IN SECTION12.3 ABOUT DOING CONTROLS. BEFORE YOUUSE NEW BOTTLES OR TUBES OF SERA,MAKE QUITE SURE THEY WORK BY TESTINGTHEM WITH RED CELLS THAT YOU KNOWARE GROUP A AND GROUP B.12.6 Cross-matching or compatibility testsAs you read earlier on, if a patient is given thewrong blood, it may kill him. For example, if groupA blood is given to a group B patient, he may die. Itis thus very important that both the donor and therecipient be carefully blood-grouped. But even this isnot enough, and so as to be quite sure that the bloodwe are going to give is safe, we do another testcalled a cross-match or a compatibility test. We crossmatchthe blood w.. are going to give to make sure itis compatible. We take a few drops of the patient’sserum and mix it 4th some of the red cells we wantto give him (some J;’ the donor’s red cells). We keepthe mixture of c&s and serum warm for about 2hours and then we look at the mixture with a microscope.When we keep something warm in this waywe say we incubate it. We incubate the donor’s cellsand the patient’s serum. If after incubation thedonor’s cells still lie evenly in the patient’s serum,then the blood is safe to give. We say the donor’scells are compatible with the patient-‘the blood iscompatible’. But, if after incubation there isagglutination, the bloqd is not safe to give thepatient. Such blood is incompatible; and if we giveit to the patient he may die. Cross-matching istherefore very important, and it must be donecarefully.There are several ways of cross-matching blood.The way we have just described uses the patient’sserum and a suspension of the donor’s cells in saline.It is therefore called the saline cross-match. A salinecross-match is useful, but an albumen cross-match isbetter. Good laboratories do both kinds of crossmatchat the same time. In an albumen cross-match adrop of 30% bovine albumen is added to a salinecross-match after the test has been set up. Albumenis one of the plasma proteins. Bovine albumen is athick liquid made from the plasma of a cow (bovinemeans from a cow). Sometimes it is bought as apowder which you dissolve in saline. If you are givenbovine albumen as a dry powder, weigh one gramand dissolve it in 3 ml of saline. Keep bovinealbumen in a small bottle with a pipette in it in thesame way as you keep anti-A serum and anti-Bserum. This is shown as Picture 9, FIGURE 12-3.Keep bovine albumen in a refrigerator. In thealbumen cross-match the donor’s red cells, thepatient’s serum and the bovine albumen are incubatedand looked at under a microscope in exactly thesame way as with the saline cross-match.The albumen cross-match, the saline cross-match,

Cross-matching or compatibility tests 1 12.6and blood grouping are all described in FIGURE 12-3.We shall describe the albumen cross-match first.METHODSTHE ALBUMEN CROSS-MATCH. FIGURE 12-31. Take a few drops of blood out of the pilot bottle ofthe donor’s blood that is to be cross-matched. You willread about the pilot bottle in Section 12.9.2. Put these red cells into a tube full ot saline. Spin(centrifuge) this tube cnf red cell suspension.3. You will be left with a small deposit of washed redcells at the bottom of the tube.4. Pipette ofF the supematant saline to leave a depositof nearly dry, washed red cells.5. Add a few drops of dean saline to the cells at thebottom of the tube. As with the previous method, onlyadd just enough saline to make a 5% suspension.6. Put two drops of the washed 5% red cell suspensioninto a small test tube. Special small test tubes forthis are in the main equipment list as ML 48b. We shallcall these small test tubes ‘cross-matching tubes’.Add two drops of the patient’s serum.7. Mix by flicking (gently hitting) the bottom of thecross-matching tube with your forefinger. Put the tubein a water-bath at 37*C. There is a water-bath (ML 53)and a thermometer (ML 44) and a thermometer sheath(ML 451 in the equipment list especially for this. Use themetal test tube rack (ML 401 to hold the tube in. Leavethe cross-matching tube in the water-bath for an hourand a half.8. Let two drops of 30% bovine albumen (9) run downthe side of the tube. Don’t mix, but incubate for a furtherhalf hour (101.After 2 hours’ incubation take a clean, empty, thickPasteur pipette, remove the cells from the bottom of thetube and lay them gently across a slide. Try not to mixthem up too much. Look at them with a low powerobjective as shown in Picture 13.THE SALINE CROSS-MATCH, FIGURE 12-3Go as far as step 5 above.Place two drops of washed cell suspension from thedonor in the cross-matching tube. Add two drops of thepatient’s serum.Mix. incubate for 2 hours and lay the deposit gentlyacross a slide. Look at the deposit with a microscope.What you may see is shown in Pictures 14, 15, and 16in FIGURE 12-3. If the blood is compatible as in Picture14 the cells will be lying separate from one another. Ifthe cells are sticking to one another (agglutinated), as inPicture 15, the blood is incompatible and ntust not begiven to the patient.Sometimes the cells will be. seen to be lying one on topof another like piles of coins in a way that is differentfrom the sticking together anyhow of agglutination.When cells lie togetlier in this way, like piles of coins,they are said to form ‘rouleaux’. (This word rouleaux isFrench for a pile of coins.) Rouleaux are shown inPicture 16 and in Picture F, FIGURE 7- 11. Blood whichforms rouleaux is safe to give to the patient. Blood cellsform rouleaux because there are special proteins in theserum of the patient.Rouleaux may also be seen because the patient hasbeen given ‘Dextran’ or something like it. ‘Dextran’ is athick clear liquid which is made in a factory. It is a kindof factory-made plasma. ‘Dextran’ is given to save the lifeof a patient who is bleeding. It is very useful instead ofblood and is often given to patients while their blood isbeing grouped and cross-matched. If there is ‘Dextran’ ina patient’s serum, it will cause rouleaux with all the bloodthat is cross-matched for him. A blood specimen forgrouping and cross-matching must therefore be takenfrom a patient before he is given ‘Dextran’. Taking ablood specimen for grouping and cross-matching issomething for a doctor or medical assistant to remember.But a laboratory assistant should know about ‘Dextran’and how it can cause rouleaux. ,It is usually easy to tell rouleaux formation fromagglutination, but sometimes it may be difficult. It is alsopossible to have both rouleaux formation and agglutinationtogether. There is another test (the Coombs test)that can be done when this happens, but we will not saymore about it here. lfyou are not sure whether you arelooking at agglutination or rouleaux, don’t give theblood.The top part of FIGURE 12-3 shows blood beinggrouped on a slide. When you group blood on a slide it isuseful to put a cross in the top left-hand corner of theslide. This cross or ‘index mark’ will help you to stopturning the slide round by mistake. If the slide is turnedround by mistake, you may mix up the drops whereyou have put anti-A and anti-B and report a wronggroup.It is always wise to re-group (group again) the bloodgroup of any bottles of stored blood as well as crossmatchingit. This is especially important if blood hasto be given in a hurry. You may well find a mistakein the ABO blood grouping more quickly with yourgrouping sera than by cross-matching. Re-groupingtakes very little time and is well worth the extratrouble.What should you do if the cross-match shows agglutinationwhich means that the blood is incompatible forthe patient? First, check the ABO group of the patientand the donor. This is the most likely cause of thetrouble. Perhaps the recipient is group A, and the donoris really group B. If this is not the cause of the troubleand the ABO groups are right, try cross-matching theblood of some other donors. The blood of one or more ofthese may be compatible. When the trouble is not just awrong ABO group (an ABO mis-match), it may be verydifficult to find out why blood is incompatible. We cannotsay more about it here. However, apart from ABOincompatibility, incompatibility is rare.

12 1 Blood TransfusionAmore thanBSET UP CONTROLSanti-A serum anti-6 serumsuspension eitherside of the slidered cells for washingMICROSCOPICAPPEARANCESOMPATIBLE 13 look at the suspension two dropswashed cclsaline method3AlI -- I - - -Plate 12piles of coins, theyare said to formrouleauxalbumenalbumenhoursthis is the bottle of bovinealbumen with a pipette and teatFig. 12-3 Cross-matchingmethod

Rhesus grouping 1 12.712.7 Rhesus grouping.JiBO blood groups are much more important thanRhesus blood groups. Even so, large laboratories alwaysgive blood of the right Rhesus group. Rhesus negativepatients should only be given Rhesus negative blood, andRhesus positive patients are usually only given Rhesuspositive blood. But Rhesus antisera for Rhesus groupingare sometimes hard to get and are expensive. Although itis possible to buy Rhesus antisera that work on a tile or aslide, most Rhesus antisera only work in a tube at 37OC(body temperature). If you are given Rhesus antisera thatwork on a slide, there will be instructions with the bottle.But you will probably be given what is called ‘albumenantisera’ or ‘albumen anti-D’. ‘D’, as you will rememberfrom Section 12.1, is another name for the most importantRhesus group. Use albumen anti-D just as if youwere doing an albumen cross-match. Use albumen anti-Dinstead of the patient’s serum. If, when you read the‘cross-match’, there is no agglutination, the patient isRhesus negative. If there is agglutination, the patient isRhesus positive. It is even more important to do controlsfor Rhesus grouping than it is to do them for ABOgrouping. For these controls you will need some red cellsthat you know are Rhesus positive and others that youknow are Rhesus negative.If you are only doing a few Rhesus groups, use EldonCards.12.8 Eldon cards (FIGURE 12- 1)These are pieces of white cardboard with a smooth surface.They are printed with squares. On one square anti-A serum has been dried. On another square anti-B serumhas been dried. Yet another is left plain. A drop of tapwater is put in each square and a drop of blood is added.The mixture is then stirred with a special plastic stick.The blood agglutinates just as it does on a tile. Somecards have another square on which anti-D serum(Rhesus serum) has been dried. These cards can be usedfor Rhesus grouping.Eldon cards are sold carefully wrapped in metal foil(‘metal paper’) and come with special instructions. Followthese instructions very carefully. Always cross-matchany blood before you give it by the albumen or salinemethod described in Section 12.6. Don’t try to crossmatchon an Eldon card, even though the card says youCm.Eldon cards are usually used with plain blood-thatis, with red cells that have not been ‘washed’. Wher theyare used like this, you may see agglutination in the controlsquare which has no antiserum. This is due to heavyrouleaux formation in the blood being grouped. Whenthis happens, wash the patient’s cells free of his serumwhich is the cause of the rouleaux. Make them up insaline in a strong (45%) suspension, just like blood. Usethis strong washed red cell suspension on the Eldon card.There should now be no agglutination in the controlsquare-the control should be negative.Because Eldon cards are sold with such good instructionsnothing more will be said about themhere. The address of the makers is given in Section13.7 (ELD).12.9 EquipmentSo far we have only talked about blood groups and howto group and cross-match blood. You must now learnabout the equipment that is used to take and store it.Many different kinds of equipment are used. We shallonly describe one kind. It isthe MRC blood transfusionequipment. MRC stands for the Medical ResearchCouncil of Great Britain who first made it. It can be usedmany times and is the cheapest kind of equipment. Somehospitals buy ready-made transfusion fluids in bottlescalled ‘Vacolitres’. Other hospitals use fluids in plasticbags and take blood into plastic bags.When a patient has not got enough blood, he mayhave to be given a litre or more of blood before he iswell again. Blood from a donor is taken into a bloodtakingbottle which holds about half a litre. If bloodwere taken into an empty bottle, it would soon clot. Wecannot give clotted blood to a patient, so we put an anticoagulantinto the bottle to stop it clotting. There aremany anticoagulant solutions, but a common one iscalled “acid citrate dextrose’ or ‘ACD’ solution. Likesequestrene, citrate stops blood clotting. Dextrose,which is another name for glucose, is food for theblood while it is being stored in the refrigerator. Thereare lG0 ml of ACD solution in the bottom of a bloodtakingbottle. When 430 ml of blood have been addedit is full.The MRC blood-taking bottle is shown in FIGURE12-4, together with the equipment that is used with it. Onthe right-hand side of this figure you will see a list of theequipment that is used with this bottle. Some of it is usedto take blood from the donor into the bottle. This is thetaking set and the airway. All the rest of the equipment isused to take blood from the bottle and put it into therecipient. This is the giving set,The bottle. Figures 12-4 and 12-5On the top of the bottle is a metal cap (1). You cannot seethis cap in FIGURE 12-4 because it is under a plasticcover or ‘Viscap’ which is described in more detail inSection 12.13. The same bottle with the ‘Viscap’ removedand the taking set in position ready for bloodtakingis shown in FIGURE 12-5. It has been put togethercarefully, so that it is still sterile.The metal cap has holes in it. through which needlescan be pushed to put in and take out blood. The bottle issealed (closed and micro-organisms kept out) by a rubberdisc or circle (2) through which needles can bepushed. It is most important that nobody should open ‘ibottle and let micro-organisms in before it is used. Thisis the purpose of the ‘Viscap’.

12 1 Blood TransfusionThe taking set and airway the guard tubes are pulled off to leave the sterile needlesready to use.These have been shown put together a! the bottom of Blood bottles are often made so that there is little orFIGURE 12-5. At the top of this figure $0~ can see the no air inside them. When there is nothing in something,pieces of the taking set and the airway taken apart. The not even air, we say there is a vacuum. Blood bottles aretaking set is made of two needles (1 Sa) on the ends of a therefore often made with a vacuum inside them. But it ispiece of rubber tube. One needle is put into the patient; difficult to make a perfect vacuum and not to leave any\1Al RWAY L- .-.J+----+a15 \1 /-I73 -3-1-aA-giving setsharpIblur 1tR88 olive..’tznthis is the ballon the cannulaI I,smcone rOr ithe tin in which to keep this equipment, and the band to goroupd the bottle are not shown in this figure1.2.3.4.5.6.7.8.9.IO.Il.12.13.14.15a.15b.16.17.THIS IS THE LIST OFALL THE EQUIPMENTCap, metal, screw thread withhole for transfusion bottle.Rubber disc, for metal screw cap.14 mmViscap, white opaque, size 6 cutdown to 2%” for use on transfusionbottle.Needle transfusion, giving 1.5 x 35mm. Olive tubing mount.MRC pattern I.V. cannula 37.5mm x 15 BWG.Adaptor male metal olive tubingmount.Tubing blood transfusion red rubber3/I 6 ” bore x ‘/ia ” wall.Tubing, blood transfusion silicone ?,a ”bore x ‘/,s ” wall.Drip counter, glass, MRC type.Clamp, regulating MRC pattern.Tube, glass 9X” for MRC giving set.Bung rubber with @NO holes MRCwe.Filter, gauze, .metal MRC type.Tubing, polyvinyl chloride 4 mmbore.Needle, blood taking, 1.9 x 40 mm.Needle, blood transfusion closure.piercing 2.1 x 37 mm.Transfusion bottle, 540 ml capacity,straight sided flint glass.Tube, glass 2%” for MRC typegiving set.all this equipment can be obtainedfrom Messrs. Turner (TUR), seeSection 13.7.Fig. 12-4 MRC blood transfusion equipmentthe other is put through the cap of the blood bottle. Only air in the nettle. If there is air in the bottle and we try toone needle on the taking set need be very sharp-the one put blood into it, the air has to come out, or the bloodwhich goes into the patient. Distinguish the sharp one by will not go in. The air must therefore be allowed to comeputting a piece of thread, wire, or a short length of bigger out as the bottle fills with blood. An airway is thereforetubing round its adaptor. Because blood goes through the used to let the air come out of the bottle. The airway is aneedles and the tube they must be sterile. The needles are needle with a shLrt piece of tube on it. In the end of thetherefore covered with short pieces of plastic tube or test short piece of tube is a piece of cotton wool. The cottontubes-these are the guard tubes. The ends of these wool is to stop micro-organisms going down theguard tubes are plugged with cotton wool. The whole tube into the bottle. The needle of the airway musttaking set is then autoclaved. Before the taking set is used be kept sterile like the needles of the taking set. It

guard tubethis rubber discgoes inside the capTAKING SET 14) Irubber tubey[)Al RWAYcotton15b rubber tube wool: QL-X-SSISWJ/ glass tubeTHIS IS THE TAKING SETREADY TO BE USEDEquipment 1 12.9into a vein on the patient’s arm. The bottle is turnedupside down, and blood flows from the bottle through thetube and needle into the patient’s vein. If blood is tocome out of the bottle air must be allowed to go into it.The long tube (11) lets air go into the bottle. It is long sothat the air can go above the blood in the bottle.There are often small clots in the blood. A wire gauzefilter (13) is therefore put over the end of the short tube(17) to stop blood clots going down this tube into thepatient. Wire gauze is a kind of metal cloth. There is ascrew clamp (tap) (10) to adjust how fast the bloodshould go into the patient. There is also a drop counter(9) to see how fast blood is flowing through the set. Thishas a wide tube with a short narrow tube inside it. Bloodfalls drop by drop from the narrow tube. By looking tosee how fast the drops are going we can see how fast theblood is flowing from the bottle into the patient. Most ofthe giving set is made of silicone tubing which lasts along time. Silicone tubing leaks (liquid comes out) after aneedle is put into it; so there is a short piece of rubbertube at the end of the giving set. Doctors can inject drugsinto the blood through this piece of rubber tube whichdoes not leak. A short piece of glass tube (17) joins thesilicone tubing to the rubber tubing.A needle cannot be fixed straight into the end of apiece of rubber tubing; so a piece of metal called anadaptor (6) is used. One end of the adaptor fits into therubber tubing and the other end fits into a special needle(4). The adaptor mustfit the needle. The round end of theadaptor which fits the tubing is called the olive.acid citrate dextrose orthis is the groove to holdthe band to hang up theFig. 12-5 The ,tiing set.iis therefore also kept covered with a guard tube until itis used.The givingsetBlood 1s given to patients in the wards by doctors andmedical assistants. Laboratory workers do not thereforeuse giving sets. But laboratory workers have to washthese sets after they have been used and make them readyfor use again.Giving sets work like this. When blood is to be givento a patient, the cap (1) is taken off the bottle and a cork(12) with two tubes (11, 17) on it is pushed into thebottle. To the shorter of these tubes some rubber tubing(8) is fixed, which goes to a needle (4). This needle is putRecordand Luer fittingsThere are two kinds of adaptor and needle: one is calledthe Luer fitting (something which fits); the other smaller,older one is called the Record fitting. These are shown inPictures R and S, FIGURE 12-6. In the same way thereare Record and Luer syringes and Record and Luerneedles for injections. A Record syringe or adaptor willnot fit a Luer needle. A Luer syringe or adaptor will notfit a Record needle. Sensible people always buy Luerfittings whenever they buy new equipment. But there isstill much Record equipment in many hospitals andclinics. Whenever you pack a needle with a syringe or anadaptor be careful to make sure that theyfit. They mustbe either both Luer or both Record. You will see thewords ‘male’ and ‘female’ with these fittings. The malefitting is the part which goes into the female fitting.The ‘cut-down’You will have been wondering what a cannula is (Picture5, FIGURE 12-4). The easiest way for a doctor or medicalassistant to give a patient a blood transfusion is to put thespecial needle (4) straight into one of the patient’s veins.But this needle is quite big, and some patients do nothave veins that are big enough for the needle to bepushed into them. A cut must therefore be made in thepatient’s skin so that a vein can be found. When a vein is

‘_:;.r. , _-

B,>Sharpening needles 1 12.10Rubber bungs. Soak the rubber bungs in detergentsolution and boil for 10 minutes. Wash them under a tapusing a test tube brush for the holes and a nail brush forthe outside. Wash away all the detergent with plenty oftap water. Wash the bungs in distilled water and leavethem to dry.Wirer gauze fiAers. Soak the filters in detergent solutionand then wash them under a running tap. Scrub theoutside with a nail brush and the inside with a test tubebrush. Gently turn back the fold at the top so that anyclots caught there can be washed away. Wash thesefilters very carefully or they will break and become us4less. Wash away the detergent with plenty of water.Hold the filter up to the light to see that it is clean andnot broken. Then wash it in distilled water and let it dry.If possible try to use these filters only once, as they aredifficult to clean.Needles. Leave the needles in detergent solution forsome hours. Clean the inside of the hub (the wide part)with a piece of wire on the end of which you havewound a piece of cotton wool. Hold the needle under thetap and run a stream of water through it. If the needle isblocked push a thin piece of wire through it. Tw not toscratch the inside of the needle or blood will clot as itgoes through on its way to the patient. Wash theneedles in distilled water. Boil them in distilled water.Rinse them in distilled water and then dry them. If theneedles are blunt. sharpen them by the method describedbelow.WHENEVER YOU USE DETERGENT, MAKE SUREIT IS WASHED AWAY WITH PLENTY OF WATERAND NOT LEFT IN THE SETS.Wash a cannula in the same way as You wash aneedle. Wash and dry the regulating clamp.You can put the sets together straight away, but ifyou leave the parts they must be kept away fromthe dust. Keep them in labelled boxes with a lidwhere cockroaches and other insects cannot get tothem.Put the sets together as shown in Figure 12-4. If theends of the tubes fit loosely, cut these ends off wherethey have become too big. When you have put a settogether, run some fresh distilled water through it andallow. it to drain. Wrap the sets carefully, using eitherthe special ‘Cellophane’ paper (‘Cellophane’ is clear,transparent paper) or brown paper. Take care that thetube is not kinked (bent sharply so that its inside isblocked). Pack each set in a tin with a needle and cannula.The doctor will use whichever he finds easiest.Make sure that you autoclave the sets within 1 hourafter you have run distilled water through them. This isimportant because if you let the sets stand withdistilled water inside the bacteria may grow, and eventhough the bacteria have been killed in the autoclavethey may harm the patient.Autoclave the sets at 15 lb. pressure for 15 minutes.Take the sets out of the autoclave and put a piece of‘Sellotape’ around the edge of the lid and the tin. Markthe tin ‘Giving Set’ and write the date they were sterilizedon the lid.PREPARING TAKING SETSPrepare taking sets in the same way as the givingsets. Make sure that the airway is not blocked by thecotton wool filter being too tight.Wrap up each set separately and take care that thetubing is not kinked. You can put up to four sets in thesame tin or you can sterilize many sets in a drum. Put‘Sellotape’ around the edge of the lid and the tin andwrite ‘Taking Sets’ on the top. Write the date of sterilizationon the tin.12.10 Sharpening needles (FIGURE 12-6)Because needles have to go into patients they must besharp. Needles are sharp when they are new, but theysoon get blunt. The best way to sharpen blunt needles isto use a special machine which has a round stone that isturned by an electric motor (a special grindstone). Butyou can easily sharpen needles with an oilstone. An oilstoneis a piece of smooth, hard stone which slowlyshapes or sharpens metal that is rubbed on it. A drop ofoil is used to carry away the little bits of metal that arerubbed off by the stone. Oilstones called ‘HandArkansas’ or ‘Washita’ are the best, but, whatever stoneyou use, it must be fine (smooth) and not coarse orrough. A new needle has a hollow concave bevel, asshown in Pictures A and B. If you look at the other sideof the needle you will see that it has two small, flat facesor facets like those shown in Picture C. You wili not beable to make a concave bevel with a flat oilstone, but youwill be able to make a flat one which will work quitewell.METHODSHARPENING A NEEDLE. FIGURE 12-6Put a drop of thin oil on your oilstone. Push the needlebackwards and forwards along the stone as in Picture L.Keep the needle in the same position as you move italong the stone. Keep the bevel the same length as on anew needle. If, as in Picturee M. you rock (tilt, tip, oralter the position of) the needle, the bevel will be roundas in Picture F. Make the bevel flat as in Picture J.When you have sharpened the needle a little you willsee a thin edge of metal on the side of the bevel as inPicture H. This is the burr. Take off the burr by gentlysharpening the needle on its outside as shown inPicture N. This will make the two facets as shown inPicture K.When you get near to the shape you want, sharpenthe needle very ge&y indeed. Always finish sharpeningby giving one rub on the bevel followed by one on each ofthe facets. When the needles have been sharpened, soakthem overnight in trichlorethylene which can be

12 1 Blood TransfusionLthere will be a flat bevel on theneedle if it is rubbed like thisthere will be a round bevel on theneedle if it is rubbed like thissee if a needle is ready to be usedNthe needle is being drawn backwardsalong the stone to produce the facetsshown in Picture K aboveLUER FITTINGTHIS IS A DIAGRAM mOF RECORD AND LUERNEEDLE FITTINGSFig. 12-6 Sharpening a needleobtained from the theatre. This will remove the oil andany small particles that may have been left inside themduring the sharpening. Drain them and polish them witha soft cloth.Before you pack a needle inject some water through‘it with a syringe, as in Picture 0. This will make surethat it is not blocked and that the hole inside it is empty.If possible look at the needle under a magnifyingglass so that you can see its shape better, as is shown inPicture P. A magnifying glass is a lens which is used forlooking at small things.Befora you pack up a needle to be sterilized, rub thepoint across your fingers, as in Picture Q. This will tellyou if it is hooked or not. Needles can be sharpened tentimes or more before they need be thrown awaybecause they are too short.The bevel must not be too short as in Picture D or toolong as in Picture E. It must not be round as in Picture For hooked as in Picture G. There must be no burr as inPicture H. A well-sharpened needle is shown in PicturesI, J, and K.These instructions have been given so that you cansharpen the needles of blood transfusion equipment. Butyou can sharpen tiny needle in the same way. Make surethat all the needles in your hospital or health centre aresharp.Once a needle has been put together with other equip-

The pilot bottle 1 12.11ment and sterilized. it must not be touched. It mustremain sterile so. as not to contaminate the patient.12.11 The pilot bottleIt is very important that no micro-organisms get into abottle of blood. If they do they may grow and. spoil theblood. If blood with micro-organisms in it is given to apatient he may die. The best way to stop microorganismsgetting into the blood is to follow this rule:NEVER OPEN THE MAIN BaTI-LE OF BLOODUNTIL THE TIME COMES TO GIVE IT TO THEPATIENT. Blood for grouping and cross-matchingshould be kept in a bijou bottle (ML 14a) containingabout 1 ml of ACD anticoagulant solution. This bottle iscalled the pilot bottle and is tied to the main bottle withwire as shown in Picture 1, FIGURE 12-3. ThepiIot bottlemust never leave the main bottle until both bottles arefinally washed up when the blood has been given to thepatient. When you want some red blood cells for groupingand cross-matching, take them from the pilot bottleand NOT from the main bottle. It does not matter ifmicro-organisms get into the pilot bottle as you open itscap to take out some red cells. But it will matter greatly ifmicro-organisms get into the main bottle.A useful way to make pilot bottles is to open one ofthe main bottles and to pipette about 1 ml of the ACDsolution inside it into several bijou bottles. (You will notbe able to use the bottle you have opened: it will becontaminated.) Autoclave these bijou bottles with theircaps on loosely. As soon as they come out of the autoclaveor pressure cooker, screw their caps down tight.When sterilized like this, ACD solution in bijou bottleswill keep for a very long time. Whenever you get bloodbottles, tie pilot bottles on to all of them with wire. If youhave no bijou bottles, use empty penicillin bottlesinstead.12.12 Taking bloodYou may be asked to take blood from a blood donor; soyou must know how to do it. Two slightly differentmethods are described: one is for bottles with a vacuum,and one is for bottles without a vacuum. If blood bottlesare made in the hospital, they will probably not contain agood vacuum.METHODTAKING BLOOD. FIGURE 12-7The donor must not suffer from giving his blood. Donot take blood from anyone who looks ill or who hasrecently been ill. If possible, measure his haemoglobinor his haematocrit to make sure he is not anaamic.Several harmful micro-organisms can go from donorto recipient through blood transfusion. The most importantof these are-the viruses causing syringe jaundicewhich have been described in Section 4.8. This is difficultto prevent, but one way to reduce the risk is neverto take blood from anyone who has been jaundiced. Askall donors if they have had jaundice, and if they havechoose another donor. Malaria can also be spread byblood transfusion, but in many countries almost everydonor will have had malaria, and they canr:ot be stoppedfrom giving blood because of this.Tie a label on to the bottle that is going to have theblood. Write the donor’s name on it, also his blood groupand the date. Write in also the date when the blood willexpire-3 weeks later (see Section 12.14). If the patienthas already been given a label, make sure it is the rightone.Ask the patient to lie down on a bed. Put a towelunder the arm which is to be bled.Put your hand round the donor’s upper arm and hold ittight. Ask him to open and shut his hand. iHis veins willswell up. Put your hand tightly round the donor’s wristand push your hand up towards his elbow. This will pushthe blood in his veins up towards his elbow and makethem swell. If there are no good veins in the first armyou look at, look at the other arm.You will want something to put round the arm tomake the veins swell up while you put the needle in. Youcan tie a piece of rubber tube around the arm as shownin Picture A. but it is better to use a sphygmomanometer(a blood pressure machine). If you use a sphygmomanometer,pump it up until it reads 50 or 60. Therubber cuff (arm band) of the sphygmomanometer shouldbe tight enough to stop the blood getting out of the armthrough the veins. But it must not be so tight thatblood cannot come into the arm through the arteries. Ifthe pressure in the cuff is right the veins of the arms willswell upThe way in which you take the blood will dependupon whether or not there is a vacuum in the bottle.IF THERE IS A VACUUM IN THE S0ITl.EChoose a large straight vein. It is better to choose onewhich does not move about too much under the skin. Inthe arm in Picture A most people would choose to putthe needle in at the place marked ‘X’. Because it iseasier to draw a needle going into a straight vein, wehave put the needle into the vein marked ‘Y‘.Clean the place over the chosen vein with a swabsoaked in ‘Hibitane’ (0.5% ‘Hibitane’ in 70% spirit). Rubthe skin well for a minute; then, using a second swabsoaked in ‘Hibitane’ solution, clean the skin over thevein from the middle outwards.Take a guard tube froan one of the needles of thetaking set. If only one of the needles is sharp, make sureyou use the sharp one. Hold the needle bevel upwardsbetween your finger and thumb. Hold it by the adaptorand NEVER touch the point or shaft of the needle withyour fingers. Hold it flat on the skin over the vein justbelow where you want to go into the vein. This is shownin Picture B. Push it through the skin. The vein may slideout of the way as you do so, as in Pictures C and D. butthis does not matter. If the vain has slid out of the way,

. ,12 1 -Blood Transfusion/simple knotin the tube vein d I , Phard togoing to gointo the vein-’ needle ofpatient’s /Fhole in the skin .&$-hole in the vein .,c> ,‘.// \ I / on top of the \ - needle has bc fentube,taking set[ \ ” forearm/&- ~~~skin~~~~~!~~..vein 7Gx.A~~..-I.,~I’..~.~~y---.2.&~r.$k%-h,,*: .-#w..%S 78 p,!J.r&..r.1..:.,,,..I,>) h>‘.. ._., .m:,..,*u . . ‘. .*‘2.- _.., _,_,_“A*-‘-*CJdt . ..+-.,, _._.. y.*,.v, .r:..s-22 1/’ \ IIDiP,“St,’these are all views from the side\/Fig. 12-7 Taking bloodput the needle back on top of the vein as in Picture E.Push the needle into the vein as in Picture F. As soon asthe needle is in the vein. hold the point of the needle upa liile, as in Pictore 0; otherwise the point of the needlewill go through the vein, and the vein will bleed into theskin. With the point of the needle held up a little so thatit does not touch the back Well of the vein, push theneedle into the vein a bii more. While you are doing thisblood will start to come down the tube.As soon as blood is coming down the tube, push theother needle of the taking set into the bottle. Blood willthen start to come down the tube very fast. Fix theneedle in the patient’s arm with a piece of adhesive tape(sticking plaster) as shown in Picture A. If the bloodstops coming move the needle gent/y in the arm. Veryoften the blood stops because the vacuum has suckedthe wall of the vein over the point of the needle. If youpress down the point of the needle, blood will probablystart coming again. If blood stops coming before thebottle is full, it may be because the vacuum is not goodenough and there is air in the bottle. Pot in an airway tolet the air out as described below. Sometimes it helps tomake the blood flow if the patient opens and closes hishand tightly round something he is holding.Gently mix the blood and the ACD solution as thebottle fills. This is very important because if you don’t dothis the blood may clot in the bottle.IF THERE IS NO VACUUM IN THE S0lX.EPull the guard tube from the needle of the airway andpush its needle into the bottle. Push one of the needlesof the taking set (the blunter one) into the bottle, holdingthe bottle firmly whi!e you do so.Put the other needle of the taking set into the vein ofthe donor’s arm, as has been described above. Don’t letthe needle of the airway touch the needle of the takingset inside the bottle; otherwise blood may go from oneneedle to the other and start coming out of the airway.WHEN THE BOlTLE IS FULL. FIGURE 12-8When the bottle is full to the bottom of the neck dothese things:

Taking blood 1 12.12this pair of forceps -.closes off the tube ofthe taking setTHIS IS THE BOTTLE OF BLOODJUST AFTER IT HAS BEEN FILLEDIN THIS PICTURETHE NEEDLE HASBEEN TAKEN OUTOF THE BLOODBOTTLE AND THEthis needle went into thepatient, notice how it isheld between the other twoneedlesthis is the pilotbottleTHE PILOTBOTTLEc there is not enoughspace in this figureto draw the wholelength of thetaking setnthe pilot bottle, the label,and the bottle of blood itsall have the same numberCblood is flowinginto the pilot bottlethis is the corkof the pilot bottleIN THIS PICTURE THE NEEDLE THAT VVENT INTOTHE PATIENT HAS BEEN PUT INTO THE PILOT BOTTLEFig. 12-8 What to do with a full bottle of blood

12 1 Blood Transfusion1. Put a pair of artery forceps on to the tub8 of thetaking set This will clamp (block or close) the tub8 ofthe taking set and stop blood flowing.2. Let down the sphygmomanometer or take off therubber tubing that has been tied round the donor’s arm.3. Put a piece of dry gauze over the needle and take itout of the donor’s arm. Put this needle b8tween theother two needles on the top of the bottle in the wayshown in Picture A. Put a piece of adhesive tape acrossthe dry gauze and ask the donor to put his thumb on itfor 5 minutes. This stops the hole in his vein bleeding.4. Take the airway out of the bottle. Put the needlewhich was in the patient’s arm in the pilot bottle. This isshown in Picture B. Take the artery forceps off the tubeof the taking set Take the needle of the taking set out ofthe bottle and hold it up in the air. The pilot bottle willfill with the blood from the tube of the taking set. This isshown in Picture C.Give the donorTHINGSTO REMEMBERa cup of tea or a cold drink.Don’t let the pressure in the cuff of a sphygmomanometerdown or remove a rubber tube until you clamp thetaking set. If you do, and the tube is not clamped, airmay go up the tube from the bottle into the donor. Thisair may kill him.Don’t take the needle out of the donor’s arm beforeyou let down the blood pressur8 cuff. If you do, thepatient may bleed into his arm. This may be painful andwill cause a bruise.Ask the patient to lie down for 15 minutes after hehas given blood. If you want his bed for another patient,ask him to lie down somewhere else. Some donors faintwhen they give blood. Before a donor faints he may sigh.look worried, and sweat may be seen on his foreheadand lips. If this happens when blood is being taken, stoptaking blood, take away the pillow, and raise the foot ofthe bed. If the donor faints afterwards, sit him downwith his head b8tween his knees.Blood donors can give blood once in 6 months. If theygive blood more often than this they may becomeanaemic.12.13 The Uganda Mobile Team (FIGURES 12-9 and12- 10)The best way to get blood for a blood bank is for amobile (moving) team from a hospital to visit places nearby where there are many donors, such as secondaryschools and offices. In this way a mobile team can bringback many pints of blood. If many donors are to be bledsafely and quickly it is important that every member ofthe team knows what he has to do, and what equipmenthe must bring with him. This section describes the workof the mobile teams in Uganda, and you may find ituseful to know what they do.In the team there is a team leader who is usually anurse, a doctor, or a medical assistant. There are also twodonor attendants, a driver, a clerk, and often a workerfrom the Red Cross. In Uganda donor attendants aretrained for blood transfusion alone, but in most countriesthe laboratory assistant will have to do this work. Theteam described here is a large one and works from ablood transfusion cemre. But a team can easily be smallerand work from a hospital. Sometimes there might onlybe a medical assistant or donor attendant and a driver.Picture A shows how the team works. Donors are firstseen by a donor attendant who measures their haemoglobin.They are then seen by a doctor or medical assistant,who makes sure they are well enough to give blood.The donors then go to a clerk who gives them a cardboardtie-on label like the one shown in Picture B. Eachdonor then takes his label and hands it to the seconddonor attendant who bleeds him. After this, he goes tosee the Red Cross worker who gives him a cup of tea or acold drink.In Uganda the donor’s haemoglobin is measured by amethod which uses copper sulphate. This method is notdescribed here, and you are advised to use the Lovibondmethod described in Section 7.1. Donors with haemoglobinof less than 12 g % should not be bled. If anyanaemic donors are found they should be told to ask fortreatment for their anaemia. Donors must also not be toosmall if they are going to give a full bottle of blood.The label shown in Picture B is a piece of cardboard5 cm x 10 cm which has been stamped with a rubberstamp and has a hole in one end. Through this hole apiece of string is threaded. The clerk fills in the detailsabout the donor on the label and takes three sticky labelsall with the same number on them. The number is thenumber of that particular pint of blood and is stamped onthe sticky labels with the paging numerator described inSection 4.3. The numerator is set to stamp the samenumber three times and then moves on to the next number.One sticky label he fixes firmly to the cardboard onthe tie-on label and the other two he only fixes loosely.One of the loose num’bered sticky labels is stuck on tothe pint of blood, and the other is stuck on to the pilotbottle. In this way it is certain that the tie-on label, thepilot bottle, and the bottle itself will a!! have the samenumber. These sticky labels are bought in rolls and arefixed to one another at their ends. Each label should be1.5cmx 5cm.Besides giving each donor a label for his bottle, theclerk does four other things. He checks the donor’s persona!record card and writes in the date of that day’sdonation. If the donor is a new one, the clerk makes out anew donor registration card. If the donor is an old oneand is giving blood at the same place in which he hasgiven it before, the clerk records the donation on theblood bank record card. The clerk keeps the blood bankrecord cards of the donors coming to each place togetherand brings these cards every time that place is revisited.For example, the cards for the National Theatre are kepttogether and brought to the National Theatre each timethe team goes there. The clerk fills in a work sheet likethat shown in Picture C. These are the records that large

The Uganda Mobile Team 1 12.13Red Cross workercold drinks hereprovidesdonor attendants bleed the patients on these bedsmeasures haemoglobinthis is the table where the driveror another donor attendant keepsthe equipment for the team -look at the next FigureTHIS IS THE LABEL FOR THE BLOOD BOTTLEthese are the three sticky labels2L\rDONORGOING OUTCOMING INthis writing is put on with Ia rubber stampBIBornE No:D0NoRhIAM&@muP A+ om=3156/7/:this label is for the pilot bottlethis label is for the blood bottle7 \I(3 ‘\with a paging numerator /string to tie the label/onto the bottleCTHIS IS THE WORK SHEET- IT LISTS ALL THE BLOOD TAKENI(D#m: 3l’f-fcy PI67 SME~ No: 1 s5lO~J &,&,!. fi&im 14.0. &u. (l&!i!i:*LAthe bottom of the worksheet has been cut offand is not shown in this pictureFig.1 2-9 The Uganda mobile blood transfusion team

12 1 Blood Transfusionblood banks use. A smaller blood bank might only needpersonal record cards for the donors and one kind ofrecord card for the blood bank.In Picture A, FIGURE 12-9, you will see the table onwhich the driver, who is also trained as a donor attendant,keeps all the blood transfusion equipment. This isshown more clearly in FIGURE 12- 10. On the left of thetable in this figure you will see the bottle of blood completewith its takiig set. With the help of the donorattendant who took the blood, the driver will empty theblood from the taking set into the pilot bottle. HE WILLALWAYS TAKE GREAT CARE TO PUT THEBLOOD FROM A PARTICULAR TAKING SETINTO THE PILOT BOTTLE BELONGING TO IT.This is very important because, if he makes a mistake,the pilot bottle and the main bottle might contain bloodof different groups. In this way blood of the wrong groupmight be given.The driver puts the used taking sets into the bucketyou see under the table. He then puts ‘Viscaps’ on top ofthe bottles. He makes quite sure that the number on thetie-on label is the same as the number on the sticky labelon the bottle of blood, and on the sticky label on the pilotbottle. Finally, he puts the full, capped, and lahelledbottles into the wire crates you see under the table on theleft. Under the table on the right you will see anotherwire crate full of empty blood bottles waiting to be filled.You will see that these empty bottles also have ‘Viscaps’.The driver is just about to take the ‘Viscap’ off an emptybottle using a paper knife. At the front of the table youwill see empty bottles waiting to be filled; they have allhad their ‘Viscaps’ removed, and on top of each of themthere is a swab soaked in ‘Hibitane’ solution. The donorattendant taking the blood will come and fetch thesebottles as he wants them.12.14 Storing blood: the blood bank (FIGURE 12- 11)Blood taken into ACD anticoagulant solution will keepfor 3 weeks in a refrigerator-not more. At the end of 3weeks it must be thrown away. A store of blood in arefrigerator is called a blood bank.There are two main kinds of refrigerator. One is theabsorption type which makes no noise and does not havea motor. The other is the compressor type which can beheard running because it has an electric motor whichdrives its compressor.The temperature inside a blood bank refrigerator mustbe very nearly the same all the time. It is not easy tomake the absorption type of refrigerator keep the sameTHIS IS THE TABLE OF EQUIPMENTSHOWN IN PICTURE A, FIGURE 12-9,this is the driver who isalso trained as a Donor Attendantthis is the bottle ofblood in Picture Ain Figure 12-8\this is the bottleof ‘Viscaps’ //fl’R))wl of ‘Hibitane’ w” Of ‘Hibdrum full of taking setctnrilmard r0zh-h~ fnr U!LJ Ithis is the crate of bottlesfilled with blood ready to betaken back to the blood bankthis is th’e bucketfor used taking setsFig. 12- 10 EquipmentIthis is a crate of empty bottlesof ACD ready to be filled with bloodthe driver has taken the ‘Viscaps’ off these bottlesand placed a ‘Hibitane’ swab on top of each ofthem : they are ready to be filled with bloodfor blood taking

I-;.~: : ,.,!r’-Storing blood: the blood bank 1 12.148’temperature all the time, but the compressor type ofrefrigerator usually does this quite easily. You must,however, keep certain rules. For example, even an expensiverefrigerator will not keep the same temperature ifpeople are always opening the door or if they leave thedoor open for a long time.A refrigerator must be kept at exactly the right temperature.The right temperature is between 4“C and 6OC.This is just a little above the temperature at which waterfreezes, which is 0°C. If blood freezes the red cells willbreak open (lyse) and the blood will be very dangerous tothe patient. It may kill him. Blood which has been storedin a refrigerator which has got too warm may also be harmful.It is very important therefore to look after a bloodbank refrigerator carefully and make sure that it alwayskeeps blood at 4OC. You must defrost the refrigeratorregularly. By defrosting we mean taking away the ice thatalways forms on the freezing part of the refrigeratorafter a few days. If this ice is not removed, the refrigeratorwill not keep blood at the right temperature.METHODDEFROSTING R BLOOD BANK REFRIGERATORIf your refrigerator works on paraffin take out the paraffintank and put out the flame.If your refrigerator works on electricity switch off theelectricity and take the plug out of the socket.lake everything out of the rafrigerator. Blood must bekept cool: so put it into another refrigerator. If you havenot got another refrigerator. or any special boxes intowhich to put blood to keep it cold, you muA try todefrost your refrigerator when it is empty or nearlyempty. Put a bucket or tray underneath the freezing partof the refrigerator (the freezer) to catch the water whichcomes from the ice as it melts. Never try to take the iceaway with a sharp instrument or tool. You may breakthe refrigerator, and it may be impossible to mend it. Ifyou want to make the ice melt quickly, put a bowl of hotwater in the refrigerator close to the ice. If you defrostthe refrigerator regularly, there should never be so muchice that it is hard to remove. The refrigerator shouldnever ‘frost up’.Leave the door open while the ice melts and falls intothe bucket or tray.Clean and dry the inside of the refrigerator.If you have an electric refrigerator, plug it in andswitch it on. If you have a paraffin refrigerator, fill up thetank with paraffin, clean the glass and trim the wick. Bytrimming we mean cutting the wick so that it is thesame height all the way along. If the wick is the sameheight, it will burn with a flame without any smoke.Put everything that you have taken out of the refrigeratorinto it again. Do not put anything back which is outof date or not wanted.The best way to record the temperature of a bloodbank is to use a special kind of recording thermometerwhich writes the temperature on a circular piece ofpaper. If you have not got a recording thermometer youcan use a ‘maximum and minimum’ thermometer whichrecords the highest and lowest temperature reached sincethe instrument was last set. If this is not available youcan use an ordinary thermometer placed i’n a blood bottlefilled with water. Look at the temperature of the waterevery day. Write it down on a piece of paper like thatshown in Picture B, FIGURE 12- 11. This has a tempera-ATHIS IS THE BLOODREFRIGERATORBANKNEVER putblood inhereB14THIS IS THE TEMPERATURE CHARTFOR THE BLOOD BANKthese are the days of the monththermoreach bottleblood hasa pilotbottleall blood isverycarefullylabelledof3temperatureindegreesCelsius1210bottle 01waterthe blood has been toocold and must be thrownaway and more donorscalled for a, j bledFig. 12-l1 The blood bank refrigerator

12 1 Blood Transfusionture written down on the left-hand side and the daysof the month along the top. You will see that the first 7days the temperature was right, and it was 4°C. Onthe eighth day someone altered the adjustment on therefrigerator. The water turned into ice, the temperaturewent below OT, and all the blood in the bloodbank had to be thrown away. New donors had tobe called for and bled. On the 13th, 14th, and 15th thetemperature was too hot. Blood does not keep wellat this temperature; it could be used, but it would bebetter to throw the blood away and get more donors andbleed them.12.15 Making blood transfusions saferA blood transfusion may be dangerous and kill thepatient because the blood is of the wrong group, becauseit was not stored well, or because the sets were not wellwashed and sterilized, or because they have become contaminatedduring storage. Blood may also be dangerousif the donor was diseased and especially if he has hadjaundice. Blood transfusions will be safer if you rememberthese instructions carefully.METHODMAKING BLOOD TRANSFUSIONS SAFERAlways label a specimen with both a patient’s namesor all his initials and if possible his hospital number aswell. Never use a bed number because patients maychange their beds!Always label any blood you have cross-matched in thesame way.Check very carefully to see that the right blood isgiven to the right patient.Don’t forget to do control tests every time you groupa donor or a recipient.Keep your stock antisera in the freezer.Always do a cross-match (compatibility test).Keep an accurate record of everything you do in theblood bank, particularly the blood you issue.Stop blood becoming contaminated by using sterileequipment and keeping it sterile. If a bottle is set up fortaking blood and not used within half an hour, don’t useit but set up a new one. If a needle is touched by mistakebefore being put into a bottle or into the oatient’s vein,don’t use it but get a new set.STORING BLOOD PROPERLYDon’t put blood’in the freezer, and don’t allow a bottleof blood to touch the freezer. It may haemolyse even if ittouches the freezer. Blood must NEVER be allowed tofreeze.Always use pilot bottles and never open a bottle ofblood until it is wanted.Check the temperature of the blood bank daily, andrecord the temperature on a graph.Don’t use blood which has been out of the blood bankrefrigerator for more than an hour.Never store blood in a ward refrigerator.Don’t use a blood bank refrigerator to store food orspecimens.Make sure that one person, and one person only, is incharge of the blood bank refrigerator.Don’t use blood unless there is a clear line betweenthe sedimented cells at the bottom of the bottle andthe supernatant plasma on top. The plasma should be apale yellow and free from any signs of haemolysis.Haemolysis is shown by a red colour which spreadsupwards from the sedimented cells at the bottom of thebottle. There is often B white layer of fat on the top ofthe plasma; this does not matter, and the blood cansafely be used.NEVER GIVE BLOOD UNLESS YOU ARESURE THAT IT IS SAFE.QUESTIONS1. Why do we wash :ed cells?2. What is an Eldon card? Why are they so useful?3. What is meant by rouleaux formation? Draw picturesto show the difference between touleaux formationand agglutination.4. What do we mean by ‘cross-matching’? What mayhappen if it is not done?5. What is ‘Dextran’ and why should we know about it?6. Draw a picture of a blood-giving set and name itsparts.7. How would you sharpen a needle? Draw pictures ofall the mistakes you should prevent when sharpening aneedle.8. What is a ‘pilot bottle’? Why do we use them?9. What are the most important things to be carefulabout when you store blood?10. What blood groups do you know? Describe oneway in which blood may be grouped.

13 1 For Pathologists, Stores Officers,and Medical Administrators(Standard English)‘Oh yes. I used to test the urine until last year when mytest tube broke.’A medical assistant in chargeof a rural health centre in adeveloping country.13.1 A standard manualThis quotation well describes the laboratory services inmany health centres, and those in district hospitals arefrequently little better. There are several reasons whyperipheral laboratory services should often be so bad.One of them is that there has hitherto been no suitabletext from which staff could learn-a deficiency that thismanual hopes to remedy. Another is that, althoughmoney is scarce, it is lrvi usuaiiy so scarce that thenecessary items of equipment costing only a few centscould not be supplied, if it was only more generallyknown what they were. The latter shortcoming thisvolume also hopes to remove and is the particular concernof this last chapter.Both the manual and its equipment list have beenprepared with the intention of saving the time and energyof those responsible for organizing pathological servicesin developing countries. Because the list and the text havebeen closely integrated with one another, two things follow.The first is that all the equipment listed here must beavailable and &cked by medical stores if the methodsthis manual describes are to be carried out. The second isthat, if this equipment is to be issued, then staff mustlearn the methods exact& as they are described here.Because of the close integration of text, methods, andequipment, it is advised most strongly that this manual.its methods, and its equipment be adopted as they stand,or with the fewest possible alterations. This is particularlyimportant where certain equipment has been extensivelyillustrated, the microscope, the balance, and thepressure cooker for example. Opinions are likely to differon certain points, and to allow for these a number ofdifferent ‘choices’ or options have been included. Theseare discussed at the end of this chapter, and all aredescribed in the text.13.2 The scope of this manualIt might seem difficult to know what to include and whatto leave out of a work of this kind. But in practice thechoice has seldom been difficult. All methods for bacteriologicalculture have been exciuded as requiring incubatorsand autoclaves, etc., which would at least doublethe cost of the laboratory. To describe them would havealso added greatly to the length of the book. Agglutinationreactions, such as those for typhoid and brucellosishave also been omitted on the grounds that they requirereagents of limited shelf life, and do not yield importantinformation with sticient economy and freqll-ncy towarrant their being undertaken in peripheral laboratories.The exclusion of a reagin test for syphilis is moredebatable. It is felt that too much reliance is often placedon this tes! i- vie*;? $ 5: kn~vn frequency of falsepositive reactions. No histological methods have beendescribed because histology is usually only done by afew specialized auxiliaries in a central laboratory. Arrangementsfor the transport of specimens to a centrallaboratory have, however. been described in detail.It might be argued, and with some reason, that themethods described here, and the outfit of laboratoryequipment that go with them, are much too rudimentary.Perhaps they are, but the fact remains that there existmany hospitals and health centres where even the simplemethods that this manual describes cannot be done.There are, for example, numerous district hospitalswhere the CSF cannot be examined, or the sugar or ureain the blood estimated. In few dispensaries or healthcentres can the haemoglobin be measured satisfactorilyor the urine tested for protein. This manual has thus beenwritten to help in raising the laboratory practice of theseinstitutions to a certain useful minimum standard.13.3 The equipment listMost of the equipment in the main list is suitable fordistrict hospitals and health centres. That suitable onlyfor hospitals is marked ‘Hospitals only’. A group ofthree capital letters inside brackets is a code for the firmssupplying a particular item. The meaning of all these

: -,._I ,a;:: ;I”,.I_ ..‘.-13 1 For Pathologists, Stores Officers, and Medical Administratorscodes is given in Section 13.7. After the code comes thefirm’s catalogue number. Thus (BTL) 215/1360/01 isBaird and Tatlock’s code number for a polythene washbottle (ML 8). A separate code number has been devisedfor all the items in the list. This starts with the letters ML(for Medical Laboratory). Thus ML 9 is a test tubebrush. It is strongly advised that, when a health serviceadopts this manual, medical stores adopt this ML codeand insert it in their catalogues. This will make it easierfor a laboratory assistant up-country to order what hewants. Some of the items listed will already be in storescatalogues, usually spread through several sections. Whenthese catalogues are reprinted it may be convenientto insert the ML numbers of these items after theirexisting code numbers, and to add new ML items towhichever section may be most suitable. For convenientreference the complete list of ML equipment and thesections in which it can be found might will be added asan appendix to such a catalogue.In many cases alternative equipment to that specifiedwould serve equally well, but, if different equipment isordered, it must agree cIoseIy with the spectjicationsprovided, if necessary amplified by the further detailsavailable in the catalogue of the maker quoted. Apparatusfor which there is no alternative supplier has beenmarked ‘NO OTHER WILL DO’. Prices have beentaken from 1971 catalogues and are given in USA dollars,converted at the rate of $2.4 to f 1 sterling (thisbeing the exchange rate that was ruling at the time the listwas made). They are mostly retail and sometimes includetax. Considerable reductions are therefore to be expectedfor bulk export orders. Equipment purchased throughUNICEF will also be substantially cheaper than theprices quoted here.13.4 Priorities in the creation of pathologicalservices-build up the peripheral units firstThe cost of the basic list of apparatus for a health centreadds up to about $450, that for a hospital to about $660.The basic chemicals for a health centre cost about $60,those for a hospital about $90. The equipment includesthe cost of an Olympus microscope, and the chemicalsare in a quantity appropriate to an initial stock. Recurrentequipment costs should be minimal and annualrecurrent expenditure on chemicals is unlikely to exceedthat of the initial stock.It is instructive to compare these figures with the totalcapital costs of hospitals and health centres. We willassume for the purposes of argument, that, after certainof the more expensive options have been chosen andtransport, etc., provided for, the cost-of the laboratoryequipment for a hospital will rise to $750, and that for ahealth centre to $500. We will also assume that ahospital has 100 beds and is built at the cost of $8,400 abed-a typical 1967 figure for East Africa. The cost ofthe laboratory equipment is seen to amount to about0.07% of the capital cost of the whole hospital. Similarly,assuming that the capital cost of a health centre issay, $25,000, then the cost of the equipment and chemicalsfor its side room amounts to only about 2% ofthis. Both these are such infinitesimally small proportionsof the total sum that to skimp on the provision of elementarylaboratory equipment must be considered totallyfalse economy.It is instructive to compare the cost of the equipmentlisted here with that provided for other laboratories. Oneregional laboratory, which would be considered comparativelymodest by the standards of industrial countries,was equipped in East Africa for the sum of $25,000.This would have provided the equipment for no less thanforty laboratories of the kind described here! It iscontended that, in the present condition of the pathologicalservices of developing countries, a given sum ofmoney is likely to produce a greater return in humanwelfare if it is used to provide many simple laboratoriesrather than a few expensive ones. In effect the choices arethese: either the blood electrolytes can be measured inone laboratory with a flame photometer (the regionallaboratory above was provided with two, the other wasspare), or the haemoglobin can be measured in fifteenhealth centres with the Lovibond disc: either one laboratorycan be equipped with an autoclave to undertakebacteriology, or ten health centres can he equipped withthe Olympus microscope to examine stools for hookwormsand blood for sickling. The moral must be toprovide cheap simple equipment first, and, except inteaching institutions, only to provide more expensiveequipment after the need for the cheap equipment hasbeen met.The equipment listed here may he cheap, but it iswanted on such a wide scale that its aggregate cost willbe substantial. All health centres, clinics. outpatientdepartments, and district hospitals need it, so do theward side rooms of larger hospitals.13.5a Some chemicals and equipment discussedSeveral methods and the equipment needed for themrequire discussion.The issue of chemicals to health centres and district hospitalsIt is strongly urged that health centres be issued withchemicals and a balance and be expected to make up alltheir own reagents. This would ease supply difficulties,and to this end full and highly simplified directions formaking every necessary reagent have been included inChapter Three. District hospital laboratories should alsobe expected to make up their own reagents except for thefew listed in Section 13.25.The distributionof chemicalsSome medical stores may prefer to buy most chemicalsin GPR grades in either barrels or drums and break themdown into smaller lots for distribution. Other medical

Upgrading peripheral laboratories 1 13.5bstores may prefer to order chemicals in plastic bottles inthe quantities suggested in Section 13.10.Many chemicals can safely be scooped out of drumsinto polythene bags for sealing with a heat sealingmachine-see FIGURE 4-3. A paper label can be putinside each bag to say what the chemical is. When firstsupplying a new laboratory these bags of chemicalsshould be accompanied by plastic bottles into whichthese chemicals can be put (such bottles have not beenincluded in the equipment list). Chemicals are cheaper inbulk, and the cost of transport and the danger of breakageis reduced both on the journey to the medical storesand on the journey from it. Most chemicals are quiteharmless and the only ones which may be difhcult todistribute in this way are ferric chloride (hygroscopic),phenol (an oily, mildly corrosive liquid at tropical temperatures),and sodium hydroxide (corrosive and deliquescent).Immersion oil and the stains are required insuch small quantities that they can well be bought insmall units. Hydrochloric and sulphuric acid should bebought and issued in Winchester quarts (2+ litres).‘Teepol’, formalin, spirit, and xylene should be bought indrums and dispensed into plastic bottles.ContainersFour types of container are listed. It is suggested that afew glass universal containers and bijou bottles be issuedfor use inside the laboratory, and that specimens be collectedin prostic po&pots and polytubes. The polypot andlid listed at ML 14c is made of polypropylene. It onlycosts $0.023 and can be boiled or autoclaved, washed,and reissued. They are thus much preferable to containersof waxed paper which cannot be autoclaved andreissued in this way, and which cost much the same.Polypots are comparatively watertight, and flat enoughfor a specimen to be looked at before a sample of it istaken for examination. They were specially developed ascontainers for pathological specimens by the Metal BoxCompany (MBO) and are highly recommended.The Olympus microscope, Model KThis has been specified, because it is cheap and beautifullymade, both optically and mechanically. It has beenillustrated in many drawings, and great pains should betaken to supply it.Some microscopes inevitably become unserviceablewith time, so medical stores should keep a stock of serviceablemicroscopes that can be exchanged with thoseneeding repair. Stocks of spare eyepieces and objectivesshould also be maintained.Fuel suppliesThe supply of suitable fuel for health centre laboratoriesis often difficult. In default of cylinders of gas or‘Labogaz’ (Choice 17, Section 13.30) issue paraflin for apressure stove and methylated spirit for a lamp.Tablet and paper tests for the blood and urineThe classical methods have been described in preferenceto the more modern and convenient ‘dip tests’ becausethey are much cheaper. Sulphosalicylic acid for testingthe urine for protein, for example, is a tenth the price of‘Albustix’. For the less commonly used tests the costdifference is less important, so ‘Acetest’ tablets have beenincluded in the main list of chemicals, and the materialsfur Rothera’s reagent included as Choice 14.Equipment for blood transfusionDetailed instructions have been given for the washingand preparation of the MRC blood transfusion equipment,which is still manufactured (TUR), although it isno longer used in the United Kingdom, having been replacedthere by plastic disposable equipment. It can alsobe used for preparing intravenous salines and is believedto be the cheapest way of giving both blood and salines inthe district hospitals of the developing countries.Rubber stamps for laboratory reportsSuggestions for suitable rubber stamps for laboratoryreports have been drawn in FIGURE 4-l. It is suggestedthat these be ordered on an appropriate scale and stockedby medical stores.13.5b Upgrading peripheral laboratoriesOne of the aims of this text is to make the upgrading ofperipheral laboratories easier, through the holding ofrefresher courses based upon it. Although he has neverhad the opportunity to try it, it is the writer’s intentionthat groups of junior staff from outlying laboratories becalled together for, say, a week, issued with a manual,given an intensive course on the methods it describes,and then sent back to their laboratories either with acomplete outfit, or else with everything that their laboratoryneeds to bring it up to the level the manual describes.The course might well be held in a secondaryschool science laboratory, and the class could be taughtwith the equipment that they were later going to takeback with them.It is not enough that medical assistants, for example,be merely given a short course and then given an o&t totake back with them. Provisiort must be made for theminimum shelving and benching shown ill FIGURE 3- 11.A project for the upgrading of a series of laboratories orhealth centres must thus include the necessary funds fortheir conversion. This should preferably also include thefitting of a laboratory sink and tap, where this isrequired, such as that in Picture F, FIGURE 3- 1(WATER STANDARDS, two way, nozzle 23 cm frombench top, (BTL) 114/3250/02, (UNICEF)one per laboratory). Such taps and a suitable sink fo;them should also be an indispensable part of any lahoratoryin a new health centre.

13.6~ TeachingThug experimental edition of this manual has been extensivelyused for teaching. It has been found important that-each student should own a copy and be taught to use it. Itis thus essential that some of the teaching sessions shouldconsist of issuing students with the equipment necessaryfor a particular method, and then requiring them to carryit out from the book. It is not easy to teach someone totzz,% -h&&i’ iiom a book, particularly at the level of thereadership intended for this one, but if an instructor cango even part of the way towards it, much of his work isdone. The writer would, incidentally, be pleased to betold of even the smallest details in which its usefulnessfor this purpose could be improved.It is hoped to prepare sets of multiple choice questionsand teaching transparencies for use with this manual.These will be very inexpensive and are to be availablefrom (TAL). The transparencies in particular may alsobe useful to the reader who merely wants to teach himself.Neither of these will be available before mid- 1974.Although primarily intended for the auxiliary, themethods described here are those with which every doctorand thus every medical student should be familiar.This means that teaching laboratories for clinical pathologyshould be equipped with the apparatus listed here,and the medical student given every opportunity tobecome proficient in the techniques described. Were thewriter responsible for such a course, he would have thestudent permitted to take this text into an examination, soas to avoid the need to commit too much to memory.Ideally such an examination should be based on multiplespecimens illustrating a hypothetical case and test theskill and speed with which a number of investigationscan be done-with the aid of the book.13.6 The supply of complete kits by UNICEFThe ideal way to buy equipment for a health centre ordistrict hospital laboratory is in the form of a completepackaged kit containing all the necessary equipment anda copy of this text. As this goes to press it is hoped thatUNICEF will be prepared to pack and distribute kits tothe specifications given here. Readers wishing to obtainsuch kits should ask their governments to requestUNICEF to pack and supply them to the governmentmedical stores. If sufficient requests are received the kitsmay eventually become a standard UNICEF item.At the time of writing a number of kits have alreadybeen supplied by Medical Assistance Programs Inc.(MAP) who may also be prepared to provide them.13.7 The addresses of suppliersHere is a list of the firms whose equipment is mentionedand the code letters, (AME) for example, that have beenused to refer to them. For convenience the cataloguenumbers of only one supplier of the common items ofequipment have been given (Baird and Tatlock), butalternative suppliers of many items may be found in thelist below. If it was a stock item at the time of writing theUNICEF number of each item or its near equivalent hasbeen included. If it was not available from UNICEF atthe time of writing, or even its near equivalent, a spacehas been left for the subsequent insertion of a UNICEFnumber.(AME) Ames Ltd., Stoke Poges, Buckinghamshire, U.K.(BDH) BDH (International) Ltd., Poole, Dorset, U.K.(BIG) H. Bickerton Ltd., Mimram House, TewinWater, Welwyn, Herts., U.K.(BTL) Baird and Tatlock (London) Ltd., FreshwaterRoad, Chadwell Heath, Essex, U.K.(CLA) Clay Adams Inc., 141 East 25th Street, NewYork, New York 100 10, U.S.A.(EAS) Eastman Kodak Ltd., Rochester, New York,U.S.A.(EEL) Evans Electroselenium Ltd., 10 1 LeadenhallStreet, London E.C.3, U.K.(ELD) Nordisk Insulin Laboratorium, Gentofte,Denmark.(EMI) James A. Jobling and Co. Ltd., E-Mil Works,Treforest Industrial Estate, Pontypridd, U.K.(GAL) Gallenkamp Ltd., Technic0 House, ChristopherStreet, London E.C.2, U.K.(G&G) Griffin and George Ltd., Ealing Road, Alperton,(GAZ)Wembley, Middx., U.K.A.D.G. Camping GAZ, 15 Rue Chateaubriand,Paris 8’, France.(GRA) Grant Instruments Ltd., Barrington, Cambridge,U.K.(GTG) George T. Gurr Ltd., 136/144 New KingsRoad, London S.W.6, U.K.(HAR) The Hartman Leddon Company, 60th Avenue,Philadelphia, PA 19143, U.S.A. The products ofthis company are retailed by The AmericanHospital Supply Corporation, InternationalDivision, 120 Raritan Centre Parkway, Edison,New Jersey, U.S.A.(HAW) Hawksley and Sons Ltd., Lancing, Sussex, U.K.(HOP) Hopkin and Williams Ltd., Freshwater Road,Chadwell Heath, Essex, U.K.(JOH)Johnsons of Hendon Ltd.. 335 Hendon Way,London N. W.4.(KEE) C. Davis Keeler Ltd., 39 Wigmore Street,London W. 1. U.K.(MAP) Medical Assistance Programs Inc., Box 50, 327Gundersen, Ilinois 60 18, U.S.A.(MBO) The Metal Box Co. (Overseas). 37 Baker Street,London W. 1. U.K.(MOS) Moseley Centrifuge Co., 119 Pentonville Road,London N. 1, U.K.(MSE) Measuring and Scientific Equipment Ltd., 25-28 Buckingham Gate, London S.W. 1, U.K.(OHA) Ohaus Scale Corporation, 29 Hanover Road,Fordham Park, New Jersey 07932, U.S.A.(OLY) Olympus Optical Co. Ltd., 43-2 Hatagaya 2-chome, Shibuya-ku, Tokyo, Japan.

,.‘. ,. .”Gelieral stores required 1 13.8a(ORT) O&IO Diagnostics, Raritan, New Jersey, U.S.A.(0X0) 0xoid Division, 0x0 Ltd., Southwark BridgeRoad, London SE. 1, U.K.(POV) Poviet Production N.V., Mauritskade 14,Amsterdam, Holland.(PRE) The Prestige Group Ltd, Prestige House, 14- 18;?Holbom, London E.C 1, U.K.(&V) British Rayophane Overseas Ltd., Lancots Lane,St. Helens, Lancashire, U.K.(TAL) TALC (Teaching aids at low cost), Institute ofChild Health, 30 Guildford Street, LondonW.C. 1, U.K.(TIN) Tintometer Ltd., Waterloo Road, Salisbury,(TUR) i?B. Turner & Co. Ltd Grecnfields RoadTindale Crescent, Bishop -Auckland, Co. Durlham, U.K.(XLO) X-lon Products Ltd., Glynn Street, LondonSE. 11, U.K.13.8a General stores requiredCertain general stores will be required. It is assumed thatthey will be available from the general issue to hospitalsand health centres. They include syringes and needles,conical urine specimen glasses, cotton wool, surgicalgauze, lysol, paraffin, and matches. These have not beenincluded in any of the lists below, nor have the rubberstamps drawn b FIGURE 4- 1. These stamps should alsobecome a medical stores item.13.8b Special equipment in the main listThis is the equipment illustrated and described inChapter Two. In most developing countries it will almostall have to be ordered from overseas.ML1 BALANCE, Ohaus, triple beam with tarebeam, sensitivity 0.1 g, capacity 6 10 g withoutattachment weights and capacity 2610 g withthese weights, (OHA) Model 760, (UNICEF)09 10502, each $49, one only.ML2 BALANCE SCOOP AND CGUNTER-WEIGHT, for use with the above balance, polypropylene,(OHA) Model 703, (UNICEF) ,each $4.8, one only.ML3 BALANCE ATTACHMENT WEIGHTS, foruse with the above balance; two 1 kg and one500 g weights bring the capacity of the balanceabove up to 2610 g, (OHA) Model 707.(UNICEF)set $10.00, one set only.This balance and’ its attachments are sufficientlysensitive for all the reagents describedin this book. It is robust and will also serve anumber of other purposes in the health centre.It is very high& recommended.ML 4 BLOOD SEDIMENTATION TUBE (Westergrensedimentation pipette), (BTL) 403/0302,(UNICEF) 0968000, each $0.6, six.ML5ML6ML7ML8ML9ML 10ML 1 IAny standard Westergren sedimentation tubewill do.BOTTLES, dropping polythene, capacity125 ml, (BTL) 215/0705, (UNICEF) 0919100will serve, each $0.17, thirty.Most of the reagents used in this book aremade up in these bottles. Bottles accurately tothis spcciflcation must be supplied. The glassequivalent will not do.BOTTLES, narrow mouth, polythene, liquidproof with cap and cone, capacity 1,000 ml,(BTL) 2 15/0480/04, (UNICEF) 09 193 10,each $0.85, eight.These are bottles for the larger volumes ofreagents, and, although locally obtained glassbottles can be used instead, there will be occasionswhen suitable ones may not be easy toget.BOTTLES, pipette, dropping, clear glass withPVC teat and ‘Polystop’ dustproof stopper,capacity 125 ml, (BTL) 215/0640/03,(UNICEF), each $0.95, five.These are for Leishman’s stain and buffer,and for saline and iodine solutions that are usedone drop at a time. Bottles accurately to thisspecification must be supplied.BOTTLES, wash, polythene, oval shape withpolythene cap and tube, capacity 250 ml, (BTL)2 15/1360/O 1, (UNICEF) 092 1200, eachS0.53, eight.Almost any polythene wash or ‘squeeze’bottle will do.BOTTLES, wide mouth, . . . six; these are describedin more detail in the ordinary equipmentas ML 62 (Section 13.9) and as oftenbeing obtainable locally. They are, however,important items, and if there is any doubt abouttheir local availability, they must be supplied.UNICEF should supply the plastic equivalent(UNICEF) 092 1300. They are used for Field’sstain and are also for holding some dryreagents.BRUSH, test tube, with winged head, overalllength 24 cm, size of head 6 x 4 cm, (BTL)2 16/00 10, (UNICEF) 0923600, each $0.05,five.Any test tube brush of about this size will do.BUNSEN BURNER, for use with bottled gas,11 mm diam., (BTL) 2 18/0426/01. (UNICEF)each S 1.6, one only.The burner supplied must be suitable for usewith bottled gas. It will only be necessary if it isdecided to supply cylinders of bottled gas (ML60) as well.STAND, for six Westergren pipettes, (BTL)403/0307, (UNICEF) 0968400, each $2.4, oneonly.This is the stand for the Westergren pipettesin ML 4.

ML 12 CENTRIFUGE, hand, four place, single speed,complete with buckets, (G&G) S 7-17-698,(UNICEF) 0926000, each $15.2, one only.This will not be needed if an electric centrifugeis supplied-see Section 13.17.ML 13a COMPARATOR, ‘Lovibond lOOO’, (TIN)DB4 10, (BTU 307/1000, (UNICEF,093 1200, each $12.6, one only. NO OTHERWILL DO.This is the best routine instrument formeasuring haemoglobin in clinics and healthcentres. It is best supplied with an adaptor totake round tubes, ML 13b (TIN) DB4 13,(UNICEF)$0.82, one only. For thetubes themselves see under ML 48e.ML 14 CONTAINERS, the following containers willbe required:(a) BOTTLE, media, McCartney, with aluminiumscrew cap and 3 mm rubber liner, capacity5 or 7 ml, also known as a ‘Bijou’ or‘miniature’ bottle, (BTL) 2 1 S/0050,(UNICEF) 0920980, each $0.06, onehundred.(b) BOTTLE, universal, with aluminium screwcap and rubber liner, wide mouth, 28 ml,(BTL) 2 1 S/0058, (UNICEF) 092 1000,each $0.08, one hundred.(c) ‘POLYPOT’, standard black 56 ml (2ounce), polypropylene tapered screw potand lid for pathological specimens, height ofpot 33 mm, diameter of lid 58 mm, (MBO), (UNICEF) 0932530 or 0932532will serve, each $0;023, order in multiplesof 1000, five hundred.(d) ‘POLYTUBE’, clear polystyrene with polythenepush-in cap, 4 ml, 38 x 13 mm,(MBO). (UNICEF)each $0.0075, order in multiples of 3,500:five hundred.The more expensive glass bottles (a) and (b)are intended for use in the laboratory. The muchcheaper ‘polypot’ (c) and ‘polytube’ (n) are forissue to the wards and patients. The polypropylene‘polypot’can be autoclaved and reissued. The‘polytube’ can be reissued after washing. Allfour t-k’pes of container are necessary. Verylargenumbersof‘polypots’are likely to be needed.ML 15 COUNTING CHAMBER, improvedNeubauer ruling, double cell, with pair of coverglasses, (BTL) 403/0040/03, (UNICEF)0948200 will serve, each $7.2, one.A single cell counting chamber can be issuedif necessary.ML 16 COVER GLASSES, for double cell countingchamber, (BTL) 403/0050, (UNICEF)0948300, pair $0.95. five pairs.These cover glasses are for the countingchamber ML 15.ML 17 COVER GLASSES, microscope, ‘cover-slips’,square 22 X 22 mm, thickness No. 2, (BTL)406/0147/33, (UNICEF) 0934001. box of 100$0.7, ten boxes.These are the ordinary thin coverslips formicroscope slides.ML 18 CYLINDERS, measuring, plastic stoppered,100 ml, graduated in 1 ml divisions, class B,(BTL) 241/l 126/07, (UNICEF) 0937430,each $2, two.This item is used for making up the majorityof stains and reagents.ML 19 CYLINDERS, graduated, polypropylene,1,000 ml, (BTL) 24 l/l 150/06, (UNICEF)09374 10, each $4.50, one.One of these is for making reagents, anothermay be needed for dichromate cleaning fluid.ML 20 DIAMOND, for writing on glass. (BTL)240/0240, (UNICEF) 0968800 will serve,each $2.80, one.This can, if necessary, be dispensed with ifglass writing pencils are supplied.ML 2 1 DISCS LOVIBOND, for use with the Lovibondcomparator listed above, (TIN) disc code numberlisted below, each $12, one disc for eachkind required. NO OTHER WILL DO.TEST (UNICEF) 0-Wcodescodes(a) Oxyhaemoglobin 5/37x(b) Sugar in blood 5/2A(c) Sugar in blood 5/2B(d) Urea in blood 5/9A(e) Urea in blood 5/9BThe oxyhaemoglobin disc should be issued toall units. The blood sugar and blood ureamethods each require two discs and will only beneeded by hospitals. The disc 5/37x has beenspecially made with low values for use in developingcountries. The disc 5/37 with highervalues will not do so well.ML 22 FILTER PAPER, Whatman No. 1, boxes ofIO0 circles: ”(a) 5.5 cm diam. (BTL)234/0290/02.:(UNICEF) ,each 80.38, five boxes.(b) 1 I cm-diam. (BTL)234/0290/05.(UNICEF) 0963000 willserve,each $0.58, five boxes.The small papers are for the solubility testfor haemoglobin S (Section 7.26). etc., and thelarger ones for filtering stains.ML 23 FILTER PUMP, plastic, lightweight, with tapconnection suitable for 12 mm bore vacuumtubing, (BTL) 235/0400, (UNICEF) 0968302,each $3.40, one only.This will only be useful if there is running

,h,,.-.ISpecialequipmentin the main list 1 13.8b”water and a suitable tap (see 13.5b and PictureF, FIGURE 3- 1).ML 24 FORCEPS, blunt points, polypropylene, 13 cmlong, (BTL) 406/0079, (UNICEF) 072 1000 or0722000 metal forceps will serve, each $1.10,tW0.These are mainly for holding slides whilestaining.ML 25 FUNNELS, polythene, flexible, (BTL)237/0200/01, (UNICEF) 0945800, 63 mmdiam., each $0.19, five.These are needed for several methods.ML 26 GAUZE, wire, tinned iron, asbestos centre,(BTL) 239/0100/02, (UNICEF) 0946500,each $0.1, one. Hospitals only.These go with the tripod.ML 28 HOLDER, for nichrome wire, with aluminiumhandle and screwed jaws, (BTL) 218/0858,(UNICEF) 0952000, each $0.84, two.This is a loopholder-see FIGURE 3-7.ML 29 LENS TISSUE, Greens No. 105, sheets30 x 20 cm, boxes of 100 sheets, (BTL)246/ 1135, (UNICEF) 0964000 will serve, box$1.35, one box.Sheets of lens tissue have been chosen inpreference to booklets as being cheaper. Theyare essential and are for cleaning microscopelenses.ML 30 (a) MICROSCOPE, monocular, (OLY)Olympus Model K, with 1~25NA Abbecondenser, mirror, plastic cover, oil bottle,blue glass filter and the following (OLY)equipment, (UNICEF), completeabout $116 ex works, one only.(b) EYEPIECE, wide field 10x, (UNICEF)each $5.50, one only.(c) OBJEC&E, 4x achromatic, (UNICEF), each $3.50, one only.(d) OBJECTIVE, 10x, achromatic, (UNICEF)each $4.40, one only.(e) OBJECTiVE, S-40x, spring loaded achromatic,(UNICEF) , each $8.80,one only.(f) OBJECTIVE, S- 100x. spring loaded achromatic,(UNICEF) , each $16.50,one only.Where electricity is available the followingilluminator should be supplied:(s) ILLUMINATOR (OLW LSK-2,(UNICEF), price ?, one only.For the above illuminator the following bulbswill be needed depending on the voltage of thearea. Supply 6 spare bulbs of the kind appropriate.Medical stores should hold many spares.Only the bulbs supplied by Olympus fit thisilluminator. There is no Philips equivalent.Some 12 volt bulbs should be stocked.(h) BULB, 110 volts, screw terminal,(UNICEF) 0960805.(i) BULB, 220 volts, screw terminal,(UNICEF) 0960905.(i) BULB, 12 volts, screw terminal, (UNICEF)(k) MECHANICAL STAGE, attachable, ungraduatedKM, (UNICEF), each!§ 11, one only.This particular microscope, the Olympusmodel K, is highIy recommended, partly becauseit is one of the best of its kind, but mainlybecause it is extensively illustrated in ChapterSix and will thus be easier for the reader of thisbook to understand. Unfortunately it has onlybeen possible to illustrate one microscope. Themicroscope (UNICEF) 0960000 will serve butis not illustrated or described here. It is theOlympus model GB which includes a mechanicalstage, so there is no need to order item (k)above. It does not, however, include an illuminator;so order either (OLY) LSK-4 whichis (UNICEF) 0960800 f&r 110 voltsor (UNICEF) 0960900 for 220 volts. Fortunately the same lamps, items (h) and (i) abovefit both the LSK-2 illuminators for the modelK and the LSK-4 illuminators for themodel GB. The LSK-2 illuminator is illustratedin FIGURE 6- 15a. The LSK-4 illuminatoris the same except that it is fitted witha different kind of clip to attach it to themicroscope.Medical stores should keep a stock of spareobjectives and eyepieces for this microscope.The replacement of a faulty objective or eyepiecemay well make a previously unserviceableinstrument usable again for comparatively littlecost. The oil immersion objectives ( x 100) andthe high power objective (x 40) are those mostlikely to need replacing.ML 3 1 PENCILS, for writing on glass, (BTL)252/01 lO/Ol, (UNICEF) 0965000, each$0.13, twenty.These are the standard grease pencils.ML 32 PIPETTE, calibrated at 0.02 ml, 0.05 ml, andO-1 ml, (EMI) G.22171 (UNICEF) ,each $0.96, six.This pipette is essential and has beenspecially designed for use with the methods describedhere.ML 33 MOUTHPIECE, to suit red and white cellpipettes, (BTL) 403/0048/ 11, (UNICEF), ten 9 1.3, two.Used with ML 32.ML 34 TUBE, rubber, 20 cm long for use with glassmouthpiece, (BTL) 403/0048/ 12, (UNICEF), ten $0.86, two.Used with ML 32.

13 For Pathologists. Stores Officers, and Medical AdministratorsML 35 PIPETTE, type one, calibrated for deliveryfrom zero mark at top to graduation mark,Class B.(a) 2 ml, (BTL) 241/2300/02, (UNICEF)0966200, each $0.84, five.(b) 5 ml, (BTL) 241/2300/03, (UNICEF)0966600, each $0.84, five.(c) 10 ml, (BTL) 241/2300/04, (UNICEF)0967000, each $0.84, five.Plastic pipettes can be supplied.ML36 PROTEINOMETER STANDARDS SET,(GAL) ME-450, (UNRZEF)$16, one only., eachHospitals only. This is only used for measuringthe CSF protein. Optional.ML 37 SLIDES, microscope, 76 mm x 25 mm, l-2-1-5 mm thick, (BTL) 406/O 155/03, (UNICEF)0969000, 100 $1.50, five hundred.The cheapest slides will serve.ML 38 SPATULA, Chattaway’s, stainless steel, 18 cm,(BTL) 260/0170/02, (UNICEF) 0969800 willserve, each $0.8, one only.ML 39 SPIRIT LAMP, stout copper and fitted withwick, 120 ml, (BTL) 1966 Catalogue2 18/ 1360, (UNICEF) 0955 100, each $4.2, oneonly.A glass spirit lamp will serve but is morebreakable.ML 40 STAND FOR TEST TUBES, anodized aluminium,to hold twenty test tubes 6 mm in diam.,(BTL) 402/0606, (UNICEF)$1.80, one only. Hospitals only., eachThis stand goes in the water bath and is forblood grouping tubes. It can be improvised.ML 4 1 STAND FOR TEST TUBES, three tiered totake two rows of tubes, rigid polythene, twelveholes 20 mm diam, (XLO) XT 3833,(UNICEF) 0978000 will serve, each $0.9,two.Exact specifications are not important.ML 42 STAND, tripod, triangular, malleable iron,(BTL) 261/1300, (UNICEF) 0985 100. each$0.90, one only. Hospitals only.Used with ML 26 for measuring the bloodsugar.ML 43 TEATS, rubber, red, (BTL) 257/0040/02,(UNICEF) 0923700, ten $0.5. ten.These must fit the glass tubing ML 5 1.ML 44 THERMOMETER, short type. general laboratorypattern, range 0°C to 50°C in l”C, about15 cm long, (BTL) 268/0266/01, (UNICEF)each $1.4, one only. Hospitals only.This ii a short thermometer for the waterbath and fits the thermometer sheath below.ML 45 THERMOMETER SHEATH, heavy brasswith cut away front, for 15 cm thermometer,(BTL) 268/0287, (UNICEF)$2.0, one only., eachThis sheath protects the thermometer ML 44above and for the water bath, ML 53.ML 46 TUBE, polypropylene, conical, plain ungraduated,nominal capacity 15 ml, (BTL)306/O 182, (UNICEF) , each $0.26,twenty.These can be replaced by the more fragileglass ones if necessary. See also ML 47.ML 47 TUBE, graduated, conical with rim, polypropylene,10 ml, (XLO) XT 5018, (UNICEF), each $0.4, five.Glass centrifuge tubes can be used, but theseplastic ones last longer. The centrifuges listedhere will take a 15 ml plain tube, but will notaccept a 15 ml graduated tube which is slightlylarger. No centrifuge tube supplied should belarger than 16 mm diam.ML 48 TUBES TEST, to the following specifications:(a) Pyrex glass, medium wall, with rim,125 x 16 mm, (BTL) 267/0045/04,(UNICEF) 0980000 will serve, ten $1.0,one hundred.Almost any standard test tube will serve.(b) Soda glass, rimless, 50 x 6 mrt, (BTL)267/0035/02, (UNICEF) , 100$1.2, one hundred.These small test tubes are for cross-matchingblood.(c) Kahn tubes, size 75 x 12 mm. rimless,(BTL) 403/0470, (UNICEF) 0979300, 100, $1.3, one hundred.Any Kahn tube will serve.(d) Lovibond cells, square section, glassmoulded, calibrated at 10 ml, (TIN) DB424. (UNICEF) pair $2.2, six.These cells are square in sdction aad are forthe ‘Lovibond lOOO’, ML 13, which has squareholes for square tubes. They are of mouldedglass and are a quarter the price of fused glasscells (TIN) , (UNICEF) 093 1204,which also fit the ‘Lovibond 1000’.(e) Some laboratories may still be using theobsolete but still serviceable type of comparatorusing round test tubes. If this is somedical stores should stock these tubes.They are-Test tubes for Lovibond comparator.13.5 mm diam., round, calibrated at10 ml, (TIN) AF 2 17, (UNICEF) 093 1245,each $0.35. For convenience in ordering.these have been given the code ML 48e.These much cheaper round test tubes can beused with the Lovibond 1000 with the use ofa moulded adaptor block, (TTN) DB 43 1,(UNICEF). $0.8. This is such acheap addition to the Lovibond comparatorthat it has been included as ML 13b.ML 49 TILE, spotting plate, white, rigid, PVC.115 x 90 x 10 mm, with twelve depressions,

,, . .ML 50ML5 1ML 52ML 53ML 54ML 55Ordinary equipment in the main list 1 13.9(BTL) 269/0050, (UNICEF), each and keys to fit the cylinder. It may be found$0.8, one only.Used mainly for blood grouping and certainurine tests (see Section 8.9).TUBING, red rubber, bore 8 mm, wall thickness3 mm, suitable for Bunsen burners, (BTL)275/0231, (UNICEF) 0988000 will serve,metre $0.9, two metres.This is for the filter pump and Bunsen burner.TUBING, soda glass, one metre lengths, ext.diam. 6-7 mm, wall thickness 0-8-0-9 mm,(BTL) 275/0060/04, (UNICEF) 0987300, kilo$1.5, two kilos. ML61For making Pasteur pipettes.WATCH GLASS, polypropylene, 80 mmdiam., (XLO) XTllOl, (UNICEF) ,each $0.48, two.ML 62These watch glasses must be plastic and arefor weighing chemicals. They can be dispensedwith and pieces of paper used instead.WATER BATH, electrical, polypropylenelined, thermostatic, 30 x 12 x 9 cm inside, withflat lid, (GRA) JB 1, (UNICEF)specify voltage, each $45, one only. Hospital;only.For blood grouping and cross matching.WICKS, for spirit lamp, (BTL) 1966 catalogue218/1420, (UNICEF) x 7, ten $0.9, ten.Spare for the spirit lamp ML 39.WIRE, nickel chrome, 22 SWG, (BTL)277/0030/02, (UNICEF) 0989000 will serve,60 g reel $1.5, one metre.Used with ML 28 for making wire loops.13.9 Ordinary equipment in the main listIn some developing countries much of this equipmentwill be locally available. Some, the pressure cooker orthe jar ML 62 for example, will probably have to beordered abroad. If outfits are being made up for areaswhere this equjyment is not readily obtainable locally, itis strongly advised that all or most of it be packed withthe special equipment specified above. Much of it isabsolute& essential.ML 56 BRUSH, paint, small watercolour, (UN1CE.F), each $0.14, two.For marking bottles and tubes.ML 57 BUCKETS, each $1.40, six. Two of themshould be galvanized (UNICEF) 2170200 andfour polythene (UNICEF) 2 170000.For washing equipment and disinfecting, etc.ML 58 CUP, polythene. domestic. (UNICEF), each $0.28, four.These are in lieu of beakers. If beakers arepreferred supply 250-ml squat form plasticbeakers as (BTL) , (UNICEF)ML 60 CYLiNDER OF BOTTLED GAS, one in useand one spare, each $4, two. Reducing valveML 63ML 64ML 65ML 66ML 67ML 68ML 69ML 70more convenient to issue small tins of bottledgas which screw straight on to a gas burner.These tins of gas are expendable which removesthe need to return and refill empty cylinders. Ifcylinders of bottled gas are not being provided,the Bunsen burner (ML lo), and the tubing redrubber (ML 5Oa), will not be needed either. Gas isuseful but not essential, and the combination of aspirit lamp (ML 39) and a parallin pressure stove(ML 71) can be used instead. Tins of bottledgas are listed in Section 13.30 as Choice 17.HAMMER (UNICEF) 4059220 or 4057000,each $1, one only.Useful for various purposes in the laboratoryand health centre.JAR, with screw cap and wide mouth, 125 ml,each $0.07, six.If these jars are not available locally, theirspecification for order abroad are: BOTTLES,wide mouth, clear glass with plastic screw capfitted with waxed disc, capacity 125 ml, (BTL)2 15/0350/05. The screw caps for these jars are(BTL) 2 15/0352/05 and are also necessary.The plastic equivalent can be supplied instead,as (UNICEF) 092 1300. These serve severalimportant purposes.OILSTONE, small, fine, preferably Washita, orhard Arkansas, (UNICEF) 0559000 or0559500, each $0.6, one only.For sharpening needles.PAINT, enamel, oil based, 120 ml jar, each$0.44, one jar of red. yellow and black,(UNICEF) 2679000 will serve, but is large.For marking tubzs. bottles, etc.PAN, enamel, 2,000 ml, preferably with twohandles, (UNICEF) 2036500 will serve, each$1, one only.For making carbol fuchsin.PEN, spirit marking, 3s ‘Magic marker’,(UNICEF) 1802808 will serve, each $0.28.three.A general purpose laboratory marker.PLASTICINE modelling clay, 250 g,(UNICEF)$0.3, one packet.This is useful for’a number of purposes; itmakes a useful drying rack for slides.PLIERS, with wire cutters, 18 cm, (UNICEF)4073500, each $1. one only.For making wire loops, etc.PRESSURE COOKER, Prestige ‘Hi-Dome’pattern, (PRE). each $11. one only.This cooker is highly preferred because it isextensively illustrated. (UNICEF) 2039500will serve. Stock spare gaskets. etc. FIGURE l-5.For sterilizing.SCISSORS, stout quality, 15 cm. (UNICEF)2270500. each $0.5. one only.For cutting paper strips, etc.

ML 71 STOVE, ‘Primus’ parall’in (kerosene), pressure,complete with ‘prickers’, spirit bottle, etc.,(UNICEF) 0 170000, each $5.60, one only.For use with pressure cooker, or in lieu ofBunsen burner for making Pasteur pipettes.13.10 Chemicals, etc.General purpose reagents are satisfactory, and analyticalgrades are not required. The quantity specified is suggestedas an initial order for 3 district hospital or healthcentre. In some countries regulations limit the use oforthotolidine on account of its carcinogenic properties. Itis used for testing stools for occult blood, and there is nosuitable al’emative method. Precautions as to its use aredescribed in the text.SOLIDSML 73ML 74ML 75ML 76ML 77ML 78ML 79ML 80ML81ML 82ML 83ACID SULPHOSALICYLIC (also known asacid, salicylsulphonic), (UNICEF) 1090000,500 g. $4.50.For urine and CSF protein.ACID TRICHLORACETIC, (UNICEF)100 g. $1.0.For Fduchet’s test for urine bilirubin.BARIUM CHLORIDE, (UNICEF) 1011965,500 g $0.90.For Fouchet’s test for urine bilirubin.BARIUM PEROXIDE, (UNICEF) 101200 1,500 g $0.95.For stool occult blood test.CUPRIC (COPPER) SULPHATE, granularcrystals, (UNICEF) 1017500, 500 g $1.20.For Benedict’s test for urine sugar.UNICEF’s ‘Benedict’s reagent ingredients kit’,(UNICEF) 1012500 replaces this item and ML87 and ML 90.FERRIC CHLORIDE HYDRATED,UNICEF) 1019000, 500 g, $0.85.For Fouchet’s test for urine bilirubin andtesting the urine for PAS.IODINE CRYSTALS, (UNICEF) 1037500,100 g %3.0.For Gram’s method, and for examiningstools for protozoa.0-TOLIDINE (not o-toluidine), (UNICEF)100 g $3.85.For s&o1 occult blood test.PHENOL, detached crystals, (UNICEF)1060000,500g $1.10.For Pandy’s test for CSF protein.p-DIMETHYL-AMINO-BENZALDEHYDE(also known as 4-dimethylamino-benzaldehyde),(UNICEF) 125 g $1.85.For Ehrlich’s test for urine urobilinogen andtest for urine sulphones.DIAMINO-ETHANE-TETRA-ACETICACID DIPOTASSIUM SALT (also known assequestric acid dipotassium salt, ethylenediaminetetra acetic acid dipotassium salt,potassium EDTA or sequestrene), (UNICEF)100 g $1.4.Anticokgulant for blood samples.ML 84 POTASSIUM FLUORIDE,250 g S 1.2.(UNICEF)For b&d sugar method.ML 85a POTASSIUM IODIDE, (UNICEF) 1067500,250 g $2.10.For Gram’s stain, and iodine solution forstools for protozoa.ML 85b POTASSIUM DIHYDROGEN PHOSPHATE(also known as potassium phosphate monobasic,anhydrous, KH,PO.,), (UNICEF)1068500,500 g $1.0.For uses, see ML 85c.ML 85~ DI-POTASSIUM HYDROGEN PHOS-PHATE (also known as potassium phosphatedibasic), anhydrous K,HPOJ, (UNICEF)5oog $1.0.Both ihese kinds of potassium phosphate(ML 85b and ML 85~) are used for makingbuffers for Leishman’s stain, for the solubilitytest for haemoglobins A and S, and for gastricwashings for AAFB.ML 85d SAPONIN WHITE, (UNICEF)$0.7.,25 gFor solubility test for haemoglobins A and S.ML 86 SODIUM ACETATE, (UNICEF)500 g $0.92.,For urine urobilinogen.ML 87 SODIUM CARBONATE ANHYDROUS,(UNICEF) 1074000, 1 kg $1.09.For Benedict’s test for urine sugar, forhaemoglobin diluting fluid, and for a modifiedRothera’s test.ML 88 SODIUM CHLORIDE. (UNICEF) 1075005,500 g, $0.85.’ For making saline for blood grouping, microscopyof stool, for form01 saline, etc.ML 90 TRI-SODIUM CITRATE,1076000, lOOOg, S2.90.(UNICEF)For Westergren ESR and Benedict’s reagentfor urine sugar.ML 91 SODIUM HYDROXIDE(UNICEF) 1078000, 500 g $0.85.PELLETS,For skin scrapings for fungi, Nessler’s solutionfor blood urea.ML 93b SODIUM DITHIONITE (also known assodium hydrosulphite), (UNICEF)500 g $0.8.7For blcod sickling test and solubility test forhaemoglobins A and S.LIQUIDSML 943 ACETIC ACID GLACIAL. (UNlCEF). 1 litre $1.2.

:~!16; ,z..: ;.,,,j, .,, ,I__ ,”FPrepared reagents 1 13.11For stool occult blood test, white cell dilutingfluid, urine protein.ML 94b ACETONE cheapest, (UNICEF)92+ litres $2.0.For drying blood pipettes; can be dispekedwith.ML 94c HYDROCHLORIC ACID CONCEN-TRATED, (UNICEF) , 2+ litres$1.2.For acid alcohol for the hot method stainingfor AAFB.ML 95 SULPHURIC ACID CONCENTRATED,(UNICEF) , 2+ litres $1.2.For the cold method of staining for AAFB(Section 3.35) and for making dichromatecleaning fluid. Can be dispensed with.ML 96 OIL FOR IMMERSION LENSES, tropicalgrade (HAW) 994950, (UNICEF) 1037000,250 ml $3.0.For microscopy-absolutely essential.ML 97 FORMALDEHYDE SOLUTION 40%,(UNICEF) 102 1000, 2+ litres $1.9.For making form01 saline.ML 98 ‘TEEPOL’ (Shell Chemicals) or other liquiddetergent, (UNICEF) , 100 ml $0.1.For cold method for staining for AAFB. Canbe dispensed with.ML 99 METHANOL (methyl alcohol) special qualityl;or preparing Leishman’s stain, (BDH), (IJNICEF) , 1 litre $2.24.For Leishman’s stain for thin blood films.Some of the cheaper grades of methanol ma-vnot be satisfactory.ML 100 SPIRIT RECTIFIED, (UNICEF),2+ litres $01.7.Cleaning the skin, cleaning slides, makingcarbol fuchsin for staining for AAFB, etc.ML101 XYLENE PURIFIED GPR, (UNICEF), 1 litre $0.97.For cleaning microscope objectivesabsolutelyessential. Also called xylol.STAINSML 102 BASIC FUCHSIN (UNICEF) 1023000,200g $3.12.For staining for AAFB, hot and coldmethods. also counterstain for Gram’smethod.ML 103 BRILLIANT CRESYL BLUE, (UNICEF)5 g $0.13.For staining blood cells for reticulocytes.ML 104 CRYSTAL VIOLET, (UNICEF) 1016500,25 g $0.70.For Gram’s method. Scnc xrieties ofmedicinal crystal violet may not be satisfactory.ML 105 FIELDS STAIN A. (BDH). (UNICEF). 25 g $1.28.ML 106 FIELDS STAIN B, (BDH), (UNICEF)25 g $1.28.For kield’s thick film method for bloodparasites, especially malaria.ML 107 MALACHITE GREEN, (UNICEF) ,25 g $0.7.As a counterstain when staining for AAFBby the hot method. Can be dispensed with ifmethylene blue (ML 108) is available.ML 108 METHYLENE BLUE, (UNICEF) 1054000,25 g $0.7.For the cold method for staining for AAFB.ML 109 UNIVERSAL INDICATOR TEST PAPER,pH 1 to 11, (UNICEF) 0932300, box ofpapers !§ 1.29.For testing the pH of the stool in lactoseintolerance, for testing the gastric juice for thepresence of free acid.ML I10 LITMUS PAPERS, (UNICEF) 0955204 redand 095206 blue, one package of each colour$0.88.For testing the pH of urine.ML 111 ‘ACETEST’ TABLETS, (AME), (UNICEF), bottle of 100 tablets $1.4.For testing the urine of diabetics for acetonebodies. If these are not supplied, sodiumnitroprusside and ammonium sulphate willhave to be supplied in lieu, as described inChoice I4 (Section 13.27).BIOLOGICALSML I 12 ANTI-A SERUM FREEZE DRIED, (POV),(STA). (UNICEF) , 6 vials $2.ML 113 ANTI-B SERUM FREEZE DRIED, (POV),(STA), (UNICEF) , 6 vials $2.ML 114a 30% BOVINE ALBUMEN FREEZEDRIED, (POV), (STA), (UNICEF) ,vial $1.4.These three items, ML I 12, 113, 1 l4a, areall used for blood grouping and will be neededby hospitals only.ML 114b GLYCEROL UREASE SOLIJTION. (HAR),(IJNICEF) , 120-ml bottle $3.60.This is for the blood urea inethod and willonly be needed if the Lovibond discs (ML 2 I dand e) and Nessler’s solution (Choice 12,Section 13.25) are also supplied. Hospitalsonly.13.11 Prepared reagentsNessler’s solution, glycerol urease, Folin and Wu’s alkalinecopper sclutiozr. phosphi?:tlo!ybdic acid solution;,10% sodium tungstate. and two-thirds normal sulphuricacid should be prepared by central or regional laborrtories,or by the medical stores, and issued to districthospitals. They are not required by health centres. Full

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I

I ,13 1 For Pathologists, Stores Officers, and Medical Admhie~eto~At least 500 additional capillary tubes will be neededwhen each instrument is supplied initially, and a largenumber should be stocked by medical stores.ML 123 (b) MICROHAEMATOCRIT CAPILLARYTUBES, low cost, (BIC), (UNICEF)0926485 will serve, gross $1.0.13.18 Choice 5. A Fuchs-Rosenthal countingchamberIf funds are available it is desirable to supply hospitalswith a double Fuchs-Rosenthal chamber O-2 mm deep(with double cover glasses to match) for examining 1h2CSF. Instructions are, however, also given for using aNeubauer chamber O-1 mm deep for the CSF.ML 124 COUNTING CHAMBER, double cellFuchs-Rosenthal ruling, (BTL) 403/0040/04,(UNICEF)each $9.4, one only.For cover glasses ;br this chamber, see ML16.13.19 Choice 6. INH in the urineIf it is considered desirable to test the urine of tuberculouspatients for INH, chloramine T and potassiumcyanide will be wanted. The chloramine T must be thebrand made bl? Eastman Kodak (EAS). The dangers ofpotassium cyanide are carefully described in the text.ML 125 POTASSIUM CYANIDE CRYSTALS,(UNICEF) 25og, $1.1.ML 126 CHLORAMINE i must be (EAS),(UNICEF) , 100 g. $0.5.13.20 Choice 7. The cyanmethaemoglobin methodThis is the most accurate method of measuring thehaemoglobin using the EEL calorimeter. It has the considerabledisadvantage in that the standards have to bekept in a refrigerator where they have a shelf life of about6 months, and that a new ampoule has to be opened eachday. It is because of these difficulties that less accuratebut more convenient standards have been included asML 117 (r) or (m) in Section 13.15.If the cyanmethaemoglobin method is to be employedthe following will be needed:ML 127 (a) CYANMETHAEMOGLOBIN STAND-DARD, for phot&metric determinations ofhaemoglobin, (BDH) 23019, (UNICEF)box of 25 x lo-ml ampoules,each box ‘$5, five boxes $These standards are probably bestordered direct by air when required andnot stocked in a central store.(b) DRABKIN’S REAGENT, conventional,stable dry reagent, (BDH) 22040,(UKICEF), box of 24 tablets.each box $5, two boxes $If these tablets are not supplied, potassiumcyanide (see Choice 6 above) andpotassium ferricyanide, (UNICEF)1066975, 100 g should be suppliedinstead.13.21 Choice 8. Sodium azide for preserving seraIf specimens of serum for serology have to travel a longway to a central laboratory for examination, it is usefulto preserve them with sodium azide.ML 128 SODIUM AZIDE, UNICEF) ,100 g $1.18.13.22 Choice 9. Ammonia for the oxyhaemoglobinmethodAlthough it is standard practice to dilute a sample ofblood in very dilute ammonia for the oxyhaemoglobinmethod, a dilute sodium carbonate solution is describedhere. Although it does not preserve the haemoglobin foras long as does ammonia, its use is advised here in healthcentres and district hospitals because ammonia is anunpleasant and hazardous substance to transport. Ifammonia is to be used the following solution should beordered. Very little will be needed.ML 129 AMMONIA SOLUTION, 22%, SG O-92,(HOP) 1402, (UNICEF) 1006000, 24 litres$1.1, lOOm1.13.24 Choice 11. Dichromate cleaning fluidThere is no provision for making up dichromate cleaningfluid in the basic list of chemicals. If it is decided to use itthe following reagent will be needed:ML 131 POTASSIUM DICHROMATE. commercial,(UNICEF) 1066960, 500 g $0.5.13.25 Choice 12. Some additional equipment andchemicals will be required if certain reagent3 arenot going to be prepared and issued by a centrallaboratoryIt is strongly advised that the following six reagents bemade and issued by a central or regional laboratory.These are: Nessler’s solution. glycerol urea. Folin andWu’s alkaline copper, phosphomolybdic acid. 10%sodium tungstate and two-thirds normal sulphuric acid.The first two are for measuring the blood urea, and thelast four are for measuring the blood sugar. Neither theblood sugar nor the blood urea are suggested on the listof techniques for health centres, so the following equipmentwill only be needed by hospitals which will not besupplied with the above six reagents.ML 134 GLYCEROL, (UNICEF) 1024500, 500 ml$1.12.

Choice 13. ‘Dextrostix’ 1 13.26ML 135 UREASE TABLETS for the determination ofurea, (BDH), (UNICEF),boxof6tablets $1.26, one box only.ML 136 MERCURIC IODIDE, (UNICEF) 1047505,200 g $4.0.ML 137 ACID, TARTARIC, (UNICEF) 1093600,5oog $1.12.ML 138 ACID, MOLYBDIC ANALAR, (UNICEF)200 g $3.0.ML 139 ACID, ~RTHOPH~~PH~RIC 89% (sGl-75), (UNICEF) , 1 litre $2.1.ML 140 SODIUM TUNGSTATE ANHYDROUS,(UNICEF) 100 g $1.3.ML 141 ACID, SULPHURI;: ANALAR, (UNICEF),500 ml $0.78.ML 142 (u) FLASK, stoppered, one mark, Class B,500 ml, (BTL) 241/1555/08, (UNICEF)0939925, each $1.2, two.(6) FLASK, stoppered, one mark, Class B,1000 ml, (BTL) 24 l! 1555/09, (UNICEF)0939930, each $1.5, two.ML 143 BEAKER, low form, Pyrex glass with spout,1000 ml, (BTL) 209/0310/10, (UNICEF)09 17000 will serve, each $0.8, two.Methods for making these reagents have not been includedin the body of the text, so they are describedbelow. Glycerol-urease can be purchased ready made(HAR) and is listed as ML 114b.tMETHODMAKING GLYCEROL-UREASE SOLUTIONTake 5 g of jack beans or else the same quantity ofjack bean meal or urease tablets. Powder the beans veryfinely in a pestle and mortar. Mix the meal or the powderedbeans well with 100 ml of 70% glycerol in distilledwater, stand overnight. centrifuge the suspension andstore the supernatant in a refrigerator-it remains activefor many months.METHODMAKING NESSLER’S SOLUTION(a) Double iodide solution. Dissolve 75 g of potassiumiodide in 50 ml distilled water. Add 100 g. of mercuriciodide, Hgl, and wait till solution is comp!ete. Thendilute to 500 ml with distMed water and filter. Dilute thefiltrate to 1,000 ml.(6) 10% sodium hydroxide. Prepare a saturated solutionof sodium hydroxide (about 55%) by adding anexcess of NaOH to about 200 ml water and stoppersecurely. After two or three days decant the clear supernatantfluid, and dilute with distilled water to 10%. (Add45 ml of water to each of 10 ml of supernatant fluid).Check the concentretian by further diluting 10 ml to25 ml, with distilled water, and titrating 10 ml of supposed4% NaOH whh N/l acid. If the concentrationdiffers from the theoretical by more than + 5% (i.e. if inthe titration 10 ml of sodium hydroxide requires morethan 10.6 ml or less than 9.5 ml of N/l acid) it must beadjusted.(c) To prepare Nessler’s reagent from the abovesolutions. Mix 350 ml of the 10% sodium hydroxide,75 ml of the double iodide and 75 ml of distilled water.Store the mixture in a dark bottle. A precipitate mayform in tim-this is of no importance, but take care notto disturb it and use only the clear supernatant.METHODPHOSPHOYOLYSDIC ACID SOLUTIONDissolve 35 g of molybdic acid and 5 g of sodiumtungstate in 200 ml of 10% NaOH plus 200 ml of waterin a litre beaker. Boil vigorously for 20-40 minutes so asto remove as completely as possible the ammoniapresent in the molybdic acid-test this by smelling thesteam from the beaker. Cool, and transfer to a 500-mlvolumetric flask, washing it with sufficient water tomake the volume 350 ml. Add 126 ml of 89% w/wphosphoric acid (S.G. 1.75) and make up to 500 ml.METHODALKALINE COPPER (FOLIN AND WU)Dissolve 40 g of anhydrous sodium carbonate inabout 400 ml of distilled water and transfer the solutionto a 1 ,OOO-ml volumetric flask. Add 7.5 g of tartaric acidand wait until this has dissolved. Then transfer quantitativelyto this flask 4.5 g of crystalline copper sulphatewhich has been dissolved in 100 ml of water. Mix andmake up to 1,000 ml. A sediment forms in time, inwhich case decant the clear supernatant solution.13.28 Choice 13. ‘Dextrostix’District hospital laboratories are likely to want tomeasure the blood sugar rather infrequently, but, whenthey do, it may be verv important. Under these circumstancesit is debatable whether Folin and Wu’s method,which is longer and more complicated, but the reagentsfor which do not deteriorate on storage, is preferable to‘Dextrostix’, which is much simpler. but which has alimited shelf life and may deteriorate rapidly if the lid isleft off the bottle in a warm climate. It may perhaps beuseful to have both methods available.ML 144 ‘DEXTROSTIX TEST PAPERS’, (AME),(UNICEF) , one bottle $3.0.13.27 Choice 14. Rothera’s test‘Acetest’ tablets (AME) have been included in the basiclist. They are slightly more expensive than the classicalRothera test, but they are used so rarely for this additionalexpense to be minimal. If the standard Rothera test

.-.,,,.; - ‘_. 2 ’,_ _ ,sf a laboratory are its manuals,and they are often lacking. It is suggested therefore13.32 A table of choicesSenior pathologists may wish to record which choicesthey recommend for use in the service for which they areresponsible. It is suggested that, if this manual is adoptedfor use in a medical service, the copies issued be markedin the table below, so that those using it shall know whichchoices it has been decided to use.District HealthChoice Hospitals CentresChoice 1: The MRC Grey wedgephotometer . . . . . . . . . .Choice 2: The EEL calorimeter . . . . . . . . . ,Choice 3: Silica gel desiccant . . . . . . . , . .Choice 4: An electric centrifuge(three alternatives listed) . . . . . . . . , ,Choice 5: A deep counting chamber . . . . . . . . . .Choice 6: Urine INH . . . . . . . . . .Choice 7: The cyanmethaemoglobinmethod *.... . . . . .Choice 8: Sodium azide for preservingsera . . . . . . . . . .Choice 9: Ammonia for the oxyhaemoglobinmethod . . . . . . . #. .Choice 10: Buffer tablets forLeishman’s stain . . . . . . . . . .Choice 11: Dichromate cleaning fluid . . . . . . . . . .Choice 12: Chemicals and equipmentsubstituting For preparedreagents . . . . . . . . . .Choice 13: ‘Dextrostix’ . . . . . . . . . .Choice 14: Rothera’s test . ...* . ..*.Choice IS: ‘Ictotest‘ tablets . . . . . .a...Chcke 16: The ‘Cellophane’ thick mm. . . . . a....Choice 17: ‘Labogaz’ . . . . . a....Choice 18: This manual . . . . . . . . . .13.33 ReferencesArcher, H. E.. and Robb, G. D. (1925) The determinationof urea, Quart. J. .‘led.. 18. 2 74.Bruce-Chwatt, L. J. (1970) De malariae morbo, Lancer,ii, 1943.Cook, G. C. ( 1967) Malabsorption, Brit. med. J., 1, 6 15.Dodu, S. A. (1967) Diabetes in the tropics, Brir. med. J.,1. 747.Eidus, L., and Hamilton, E. J. (1964) A new method forthe determination of N-acetyl isoniazide in the urineof ambulatory patients, Amer. Rev. resp. Dis., 89, 587.

p... ‘,( I’ ‘8a..:g& >,&Ii II: /, L /_ ,, .., , , .i “.,- .- J, I : , : :,.‘:_,P~.I~‘:_ I ,I, ,; >~f~,~ _I I,_ ,/‘,:,z,;:‘:‘i,.;C _ $9 ’-1: ‘.;‘References 1 13.33Folin, O., and Wu, H. (1920) The determination of blood methode de coloration rapide . . ., Rev. Tuberc. (Paris),sugar, L biol. Chem., 41, 367.24, 1044.; Huntsman, R. G., Barclay, G. P. T., Canning, D. M., and Ritchie, L. S. (1948) An ether sedimentation techniqueCawson, G. I. (1970) A rapid whole blood solubility for routine stool examination, Bull. U.S. Army med.test to differentiate the sickle cell trait from sickle Dep., 8, 326.cell anaemia, J. din. Path., 23, 781.Sundeman, F. W., et al. (1935) Symposium on clinicalKing, E. J. (1948) The determination of haemoglobin, -haemoglobinometry, Amer. J. clin. Path., 23, 5 19.‘L&vet, ii, 563.Wld Hlth Org. techn. Rep. Ser. (1967) Control ofKing, E. J. (1948) The determination of haemoglobin,Lance& ii, 971.Lapeyssonnie, L., and Causse, C. (1960) Note sur uneascariasis, 379, 41.WId HIth Org. techn. Rep. Ser. (1969) Amoebiasis, 421,34.EpilogueThe opportunity of a spare page on which to insert anepilogue gives me the chance of leaving some finalmessages with the reader after proofs have beencorrected, and before the book finally goes to press.First, I hope that the careful specification of supplierswill not limit the use of this text. If you can find cheaper,better, or more easily obtainable equipment of the samespecifications as those listed here, buy it. The cataloguenumbers of individual firms have only been included inthe hope of specifying equipment more exactly, andbetter sources of supply may be found than thosesuggested here.New equipment is under development, and before bulkpurchases are made, ask the Tintometer company (TIN)for details of their new haemoglobinometer whichpromises to be better suited to health centre use than the‘Lovibond 1000’ listed here.The recent price reduction for plastic disposable bloodtransfusion equipment has made the rubber re-usableequipment suggested here much less competitive and itshould probably now be considered obsolete.Finally, before saying once more how much I lookforward to improving a further edition---should this onesell out-1 should like to thank the staff of OxfordUniversity Press, particularly Mr. A. E. Gray, both forthe care he has taken with this book, and for the kindnessand forbearance with which he has borne the author’simpatience.July 1973 MAURICE KING

Vocabulary IndexdYAAAFB. Acid and alcohol fast bacilli, 3.12,9.18, 11.1abnormal. If something is only found in sick people, wesay it is abnormal, 1.3ABO blood groups. The most important different kindsof blood, 12.1AR0 incompatibility. Blood which is not compatible fora patient because it is the wrong ABO group, 12.1absorbed. Soaked up, held up, 5.12accurate. Exact, 1.3ACD solution. An anticoagulant solution used in bloodtransfusion, 12.9acetic acid (ML 94a). A colourless liquid with a strongsmell used to test stools for occult blood, 10.11, fortest’ng the urine for protein, 8.3, and for making whitecell diluting fluid, 3.45‘Acetest’ tablets (ML 111). Used for testing the bloodand urine for acetone, 2.4, 7.43, 8.7acetone. A substance found in the blood and the urine ofpatients with severe diabetes, 7.43, 8.5, 8.7, 13.10acid. See 1.6acid alcohol. A reagent used in the Ziehl-Neelsen.method, 3.16, 11.1acute. An acute disease is a short-lasting severe disease,1.3adaptor. The part of a needle that fits on to a syringe,9.5, 12.9addresses of equipment suppliers. 13.1adjust. To tix or alter something so that it works better,1.3adult. A fully grown organism, 1.3‘Afriga’. A kind of bottled gas 3.4agglutination. The sticking together of particles, such asred cells, by antibodies, 12.1albumen. A plasma protein which is used in the albumencross-match, 12.6‘A~co~Q~‘. A drug used in treating hookworm infection.The same as ‘Bephenium’, 7.6alkali. See 1.6Qluminium. A soft light metalammonia (ML 129). A dangerous liquid with a verystrong smell, 1.10, 3.31, 7.1, 7.41amoeba. A protozoon which moves with the help of ‘feet’or pseudopodia, 1.9, 1.14, 10.7amoebic dysentery. Dysentery due to Entamoebahistok’ytica, 10.7amorphous. Without shape, 8.13Qmpoule. A small bottle for drugsQnQemia. An anaemic patient has too little haemoglobinin his blood, 5.9, 7.5, 7.28Anc?vtostoma duodenale. One of the hookworms, 10.5ancylostomiasis. A disease due to infection of the gutwith the hookworm, 1.12, 7.6, 10.5anhydrous. Dry, without water, 2.4Qnisocytosis. Unequally sized red cells, 7.19anti-A and anti-B serum (ML I 12, ML 113). These arcantisera used in blood grouping, 2.4, 12.3, 13.10antibodies. Special proteins in the blood which can combinewith a group of substances called antigens, 12.1anticoagulant. A chemical, such as sequestrene, whichstops blood clotting, 1.17antigens. Substances which, when they get into the body,cause the body to make antibodies against them, 12.1antiseptic. A chemical solution, such as iodine, for killingmicro-organisms on the surface of the body, 1.19, 9.5antisera. Sera containing antibodies, 12.3anuriu. A patient who makes no urine suffers fromanuria, 12.2anus. The hole through which stools leave the body, 10.4aplastic. When the marrow stops making blood cells it issaid to be aplastic, 7.27aarachnoid mater. The net-like middle covering of thebrain, 9.1area. Part of something flat, such as part of a country ora town or a field, or the skin, or a blood film, etc.arteries. Thick-walled blood vessels taking blood fromthe heart to the tissues, 4.7asbestos. A special kind of wool which cannot be burnt.It is mined from the earth, 2.2, 11.1AscQris lumbricoides. A long, thin, round, white wormwhich lives in the gut, 10.1, 10.5-ase. Most enzymes end in -ase(lactase, urease)aseptic. Without infection, 1.22aseptic precautions. To do something using aseptic precautionsis to do it while taking care that no microorganismsget where they are not wanted, 1.22aspirator jar. A big jar with a tap at the bottom, 3.3atypical. Unusual, extraordinary, 1.3

VocabularyIndexautoclave. A machine for killing micro-organisms andmaking things sterile using steam at a high temperatureand pressure, 1.20cuuospermia. No spermatozoa in the seminal fluid, 11.10BbQdQry drsentery. Dysentery due to bacilli of thegenus Shigella, i0.8bacittus, bQciZZi. A rod-shaped bacterium, 1.14Bacittus Qnthracis. A big Gram-positive bacillus whichcauses a disease called anthrax, 11.7bacteriit meningitis. Meningitis caused by bacteria, 9.16bacteriology. The study of bacteria, 4.10bactfrium, bacteria A small one-celled micro-organismin which there is no separate nucleus, 1.14balance. ‘A machine for weighing (ML 1), 5.1barium chloride (ML 75). A white powder which is dissolvedto make a 10% solution, used in Fouchet’s test,2.4, 3.17, 8.8, 13.10barium peroxide (ML 76). A white powder used for theoccult blood test, 2.4, 3.35, 10.10barrel. The outer part of a syringe, FIGURE 4-3brrsic fuchsin (ML 102). Deep purple crystals used tomake carbol fuchsin stain, 3.23, 11.1, 13.10busophil. Staining with basic stains. A basophil polymorphis an uncommon kind of polymorph with largeblue granules in its cytoplasm, 7.14, Picture D,FIGURE 7-9batter?,, A ‘box’ for storing electricity, 3.7beaker. A laboratory cup. 3.11beam. The main part of a balance which swings, 5.1bench. A laboratory table, 3.1, 3.46Benedict’s reagent or solution. A reagent used for testingthe urine for sugar, 3.18, 8.3bephenium. A drug used for treating hookworm infection,7.6bevel. The sloping edge or end of something, 12.10bijou bottle (ML 14a). A small specimen bottle with ascrew cap, 2.2,4.6bile. A thick yellow-green fluid made by the liver andexcreted into the intestine, 8.8bilirubin. One of the pigments in the bile, 8.8binocular. Two eyed. A microscope with two eyepieces,6.8biochemistry. The chemistry of living organisms, 4.10blank. A ‘blank’ tube or cell is one filled with plain wateronly, 5.18block. A square piece of something, 4.10blood, methods for. Chapter Sevenblood grouping. Finding someone’s blood group, 12.5blood groups. The different kinds of blood, 12.1blood sedimentation tube (ML 4). A tube used for measuringthe ESR, 7.39blood sugar. See 7.42, 8.6blood transfusion. Taking blood from one person andgiving it to someone else, Chapter Twelveblood ureQ. See 7.41‘bloody tap’. Blood may get into the CSF during a diffi-cult lumbar puncture. This is often called a ‘bloodytap’, 9.9Borreliu duttoni. The organism causing relapsing fever,7.35bovine. From a cow, 12.6bovine albumen (ML 114). One of the plasma proteins ofa cow used in blood transfusions, 12.6, 13.10box. A place on a form in which something is written,3.11brilliant cresyl blue (ML 103). Deep blue crystals usedfor staining reticulocytes, 2.4, 3.19, 7.23Brownian movement. A kind of movement shown by anyvery small particle lying free in a liquid, 8.14bruise. A coloured mark made by blood in the tissues,12.12buckets. The parts of a centrifuge which hold the centrifugetubes, 1.5bufir. A mixture of salts that helps to keep the pH of asolution the same, 1.7, 3.20, 3.21, 7.12bulb. (i) A hollow glass ball. (ii) The glass from whichelectric light comesbung. A rubber ‘cork’, 12.9Bunsen burner (ML 10). A special stove or burner forburning gas, 2.2, 3.4burr. The thin edge of metal on a badly sharpened tool orneedle, 12.10buttocks. The parts of our bodies that we sit on, 11.1 lbCcannub. A blunt tube that is put into a patient’s vein, 12.9capillaries. Very small blood vessels joining arteries toveins, 4.7capillary blood. Blood taken from capillaries, 4.7carbot fuchsin. A stain made of phenol (carbolic acid)and fuchsin used for the Ziehl-Neelsen method. Thereis a ‘hot’ and a ‘cold’ type of stain, 3.22b, 3.23, 11.1.There is also dilute carbol fuchsin used with Gram’smethod, 3.24case. A patient with a particular disease is said to be acase of that disease, 1.3casts. Special things found in the urine, 8.13catatogue. A book describing equipment, 2.1catgut. A kind of surgical ‘string’ made from the guts ofanimals, 12.9caustic. Burning, 1.6cell. (i) The ‘bricks’ from which the bodies of largeorganisms are made, 1.9. (ii) A carefully made glassbox used for holding coloured solutions, 5.11. (iii)The parts from which an electric battery is made, 3.7.(iv) A selenium cell is a piece of metal which makeselectricity when light falls on it, 5.16cell membrane. The outer ‘coat’ of a cell, 1.9‘Cellophane’. Thin transparent ‘paper’, 10.2b, 12.9cent. A hundred or a hundredth partcentering screws. Part of a condenser, 6.4CentigrQde. A scale for measuring temperature in whichwater boils at 100, and freezes at 0. On the Centigradescale the temperature of a normal human body is.-37°C

VocabularyIndexcentral laboratory. Sending specimens to a centrallaboratory, 4.10centrifuse (ML 12). A machine for turning tubes of asuspension round very fast, 1.5, 2.5, 13.7centrz$xge tube (ML 46 and 47). Special tubes which fitinto the buckets of a centrifuge. 1.5. 2.2. 5.7cerebral malaria. Malaria of the brain, 7.32, 9.20cerebi*ospinal jluid. The fluid that washes round thebrain. Chapter Ninecestode. A tapeworm is a cestode, 1.14chemical. A chemical is a pure substance, 1.4, 13.10chip. A small piece cut off something such as a glasschip, 4.7chloroquine. A drug used to treat malaria, 7.34choices. These are other chemicals and equipment youmight use and which are not in the main lists, 2.5,13.12, 13.13, etc.chromatin. Coloured material inside the nucleus, 1.9,10.7chromatin dot. Part of a malarial trophozoite, 7.32chronic. A chronic disease is a -long-iastiiigdisease,1.3chuck. The part of a loop holder into which the wire fits,3.10clamp. Something for holding things with, 12.9clear. Easily seen through, 1.3click stop. A catch which makes the objectives on therevolving nosepiece of a microscope stop exactlyunder the tube, 6.7‘Clinitest’ tablets. Tablets for testing the stool or urinefor sugar, 10.11Clonorchis sinensis. See 10.6clot. The soft red solid which forms when blood isallowed to stand, 1.17coarse. Rough, thick, largecoarse adjustment. A knob which causes large movementsof the tube or stage of a microscope, 6.9coaxial. Two knobs or wheels are coaxial when they bothmove on the same rod or axle, 6.9coccus, cocci. A round bacterium like a ball, 1.14cockroach. A big insect that lives in housescollar. Something which goes round something else, 3.4,3.5coma. A patient in coma seems to bc asleep but cannot bewoken up, 8.6comparator (ML 13). An instrument for comparingthings, 5.10compartment. A space or room, 5.14compatible. Donor blood is compatible for a patient ifthere is no agglutination when it is mixed andincubated with his serum; compatible blood is safeblood, 12.6concave. Hollow, empty, 6.3, 12.10concentrated. A concentrated solution is one in whichthere is a lot of something dissolved. A strong solution,1.4concentration. The concentration of a solution is theamount of something that is dissolved in itconcentration method, Methods which concentrate orgather together the parasites in a specimen, 7.38,10.3condenser. Part of a microscope containing severa;lenses which shines light on the thing we are lookingat (the object), 6.2conical. Shaped like a cone, FIGURE l-2contacts. (i) The contacts of a patient with an infectiousdisease include the person who infected him and thepeople whom he infected, 11.4a. (ii) Pieces of metalcarrying electricity, 5.25container (ML 14). A bottle or box for putting specimensin, 2.2, 4.6, 13.8bcontaminate. To contaminate something is to let microorganisms(or anything else which might be harmful)get to itcontract. To get smallercontrast. Specimens of poor contrast are those likeunstained cells which can be difficult to see with amicroscope, 6.15control valve. A ‘tap’ on a pressure Looker which controlsthe pressure of steam inside it, 1.21controls. Tests you do to see if a method is working,1.24, 7.26, 12.3copper sulphate (ML 77). Blue crystals used for makingBenedict’s reagent, 2.4, 3.18, 8.3, 8.6, 13.10cotton wool. A kind of clean soft white cotton used inhospitals, 3.9, 13.8acounterstain. A stain used to make cells or tissues adifferent colour from whatever is being stained by themain stain, 11.1counting chamber (ML 15). A special piece of glass forcounting cells, etc., 2.2, 2.5, 3.12, 7.29cover glass (ML 16). A thick glass square that fits on topof a counting chamber, 2.2, 7.29, 13.8bcoverslip (ML 17). A very thin glass square that is put ontop of a wet specimen on a slide, 2.2, 3.12, 6.7, 13.8bcrenated. Shrivelled up, 1.18, FIGURE 7- 15, 7.25, 8.13crisis. A time during an illness when the patient becomesvery ill indeed, 7.27across infection. An infection which goes from one patientto another in a hospital or health centre, 4.8cross-matching. A way of telling if a donor’s blood is safeto give to a patient, 12.6crystals. Pieces of a solid which have the same simpleshape, such as pieces of salt or sugar, 1.4crystal violet (ML 104). Deep violet crystals used inGram’s method, 2.4, 3.25, 11.5, 13.10CSF. Cerebrospinal fluid, 9.1CSF protein. See 9.13‘cut down’. When a needle is put into a patient’s vein bydoing a small surgical operation, this is called a ‘cutdown’, 12.9cyanide. Potassium cyanide is a very dangerous chemical,8.9, 13.19cylinder. (i) See measuring cylinder. (ii) A strong steelbottle containing gas under high pressurecyst. The smaller ‘sleeping’ form of a protozoon, 1.14,10.6, 10.9, 10.10-cyte. Words ending in ‘cyte’ mean some kind of cell

Vocabulary Indexcy~toplasm. IIe complicated watery mixture of things(especially proteins) inside cells, 1.9Ddash board. The part of a car where the instruments are,3.7DDS. One of the sulphone drugs, 8.10debris. Dirt, waste, rubbish, the remains of anything destroyed,7.3 1decimal. A way of writing fractions as tenths, htmdredths,etc., 5.1decimal point. The ‘dot’ in the middle of a decimal, 5.1deficiency anaemia. An anaemia due to lack of thethings that the body needs to make blood, 7.5, 7.6,7.7, 7.8defrosting. Removing the ice from the freezer of a refrigerator,12.14dehydrated. Lacking water, 8.6deposit. The particles at the bottom of a liquid, 1.4detergent. A very strong kind of soap, 1.3‘Dextran’. A thick fluid which is given to a patient beforethere is time to give him a blood transfusion, 12.6diabetes, diabetic. A disease in which there is sugar in theurine, 7.42, 7.43, 8.5,8.6diagnosis. The name of the disease that a patient has. Todiagnose a patient is to find what disease he has(Second Preface)diagram. A simple drawing in which only the mostimportant things are shown, 1.1diamond pencil (ML 20). A pencil with a very hard stonecalled a diamond in it for writing on glass, 2.2, 13.8bdichromate cleaning fluid. A solution of potassium dichromateand sulphuric acid used for cleaning verydirty glassware, 2.5, 3.12. 3.26, 13.24dt@erential white count. A count of the different kinds ofwhite cells in the blood, 7.16, 7.22dilute. A dilute solution is one in which there is a little ofsomething dissolved, 1.4Dipetalonema perstans. A filarial worm, 7.37Diphyllobothrium latum. See 10.6diplococci. Cocci which are seen in pairs, 11.6disc. A circle of something, a plate is a disc, 5.10discharge. An abnormal Auid, especially pus, comingfrom some part of the body, l-3,8.4, 11.6disinfectant. A strong chemical solution such as lysolwhich is used for killing micro-organisms outside thebody, 1.19, 3.12, 3.46dissolve. When a solid seems to disappear in a liquid, likesugar in tea, it is said to dissolve, 1.3distilled water. A specially pure kind of water, 3.15, 12.9donor. A donor is someone who gives blood, 12.1drill. A tool for making holes, 3.11dropping bottle (ML 5). A special bottle for letting reagentsfall out drop by drop, 2.2, 13.8bduct. A tube, 8.13dura mater. The tough outer covering of the brain, 9.1dysentery. Bad diarrhoea with blood in the stools, 10.8dysuria. Pain on passing urine, 8.4EEconomics of laboratory services. See 13.4EEL. A short way of writing EEL calorimeter, 5.16EEL Colorimeter (ML 117). A machine for measuringcoloured solutions, 2.5, 5.16 to 5.25, 7.41, 7.42,13.15EEL tube. A special tube which is only used with theEEL Colorimeter, 5.16, Picture C, FIGURE 5-8Eldon cards. Special cards used for grouping blood, 12.8electricity. See 3.6elephantiasis. A disease in which parts of the body swellso that they look like the parts of an elephant, 7.37Entamoeba coli. A harmless amoeba which lives in thegut, 10.7Entamoeba histolytica. An amoeba which causesamoebic dysentery, 1.14, 10.7Ehrlich’s method and reagent. A test and reagent forurobilinogen, 8.8Enterobius vermicularis. The thread worm, 10.4enzymes. Special proteins that make the things cells need,1.10eosinophil. Staining with eosin. Eosin is an orange stainin Leishman’s stain. An ‘eosinophil’ is a polymorphwith large orange staining granules in its cytoplasm,7.14. Picture J, FIGURE 7-9eosinophilia. An abnormally high number of eosinophilpolymorphs in the blood, 7.2 1epithelial cell. A cell from the epithelium, 1.9, 8.11, 8.13epithefium. The layer of cells covering the outside orinside of an organ or part of the body, 1.9equipment. Special tools or machines (Second Preface)equipment list. See 13.3, 13.8bequipment for transfusion. See 12.9eradicate. To drive out or stamp out, 7.33erythrocyte. Another name for a red blood cell; 1.9Escherichia coli. A bacterium which is a common causeof urinary infection, 8.13ESR. Erythrocyte sedimentation rate or blood sedimentationrate, 7.39ethanol or ethyl alcohol (ML 100). This is ‘ordinaryalcohol’ or spirit, 2.4evaporate. When a liquid goes into the air it is said toevaporate. 1.4examination. ‘Giving a patient a medical examination,means looking at him to see what is wrong with him(Second Preface)expand. To get biggerexudate. An abnormal fluid formed by a tissue, this iscommonly pus, 1.3, 7.14eyepiece. The part of an instrument which goes next toyour eye and through which you look, 5.11, 6.1,6.8facet. A small face, or surfa:e, 12.10faeces. The waste from the bowel or gut, Chapter Tenfalse positive report. A report which is sent out positivewhen it should be negative, 11.2Fasciolopsis buski. See 10.6fast. Able to hold on to a stain, 11.1

Vocabulary Index . .female. A woman or belonging to a womanferric chloride (ML 78). Dark brown crystals used tomake Fouchet’s reagent, 2.4, 3.30, 8.8, 8.9field of view. The view through the eyepiece of an instrument,6.7Field’s method. stains A and B (ML 105, ML 106). Ared and a blue powder used for staining thick bloodfilms, 2.4, 3.28. 7.32filariac. Nematode worms which live in the tissues, 7.37Jilariasis. The disease caused by ftlariae, 7.37file. (i) A tool for shaping something, 3.9. (li) A book ordrawer to put papers in, 4.1film. Something spread very thinly. Here it means a specimen,such as blood, spread very thinly on a slide, 1.3filter. (i) To remove the particles from a liquid by pouringit through something, usually a special paper,which will let the liquid go through and hold theparticles back, 1.5. (ii) A piece of coloured glasswhich only lets light of certain cdours go through,5.12,6.5firter paper (ML 22). Circles of a special soft paper forfiltering liquids, 2.2, 3.11, 13.8bfilter pump (ML 23). An instrument for sucking airwhich fits on to a tap and uses a stream of water to dothe sucking, 2.2, 3.3, 13.8b#Itrate. The clear liquid which passes through a filterpaper, 1.5fine. Smooth, thin, smallJine a@&rnent. A knob which causes small movementsof the tube or stage of a microscope, 6.9fine aeustment gauge. Three lines on the side of a mictoscopeto tell us how near the top or bottom of its run thefine adjustment is, 6.12Jix, fixation, fixative. To fix a tissue is to kill it and tokeep it looking just as it did when it was alive, 4.10,7.12. 11.1flagellate. A protozoon which moves with the help offlagella, 10.10, FIGURE lO- 13Jlagellum,jlagella. The hairs on a micro-organism whichmove and so move the organism, 1.14flame. To flame something, such as a loop, a scalpel or aPasteur pipette, is to kill the micro-organisms on it byputting it through a flame, I.20,3. iiiflask. A thin glass bottle used in a laboratory, 4.10fluid. In this book a fluid is used to mean the same thing as aliquid, 1.4focal length. The distance of an object from the bottomlens of an objective, at which the object is clearly infocus, 6.7focus. To focus an instrument is to adjust it so that you cansee something through it clearly, 5.14,6.7folic acid. A substance in the food, lack or deficiency ofwhich causes a special kind of anaemia, 7.7fontanelles. The soft parts of an infant’s skull, 9.3for;rL(3M8\ 24). An instrument for holding things with,.jortna~dehyde or formaiin (ML 97). This is a strongsmelling liquid used to make form01 saline for fixingtissues for histology, 2.4,3.29,4.10,13.10formed. Having some shape, 10.1form01 ether concentration test. A way of concentratingparasites in the stool, 10.3form01 saline. A solution of formalin and salt used forpreserving or fixing tissues, 1.18,4.10Fouchet’s method and reagent. A test for bilirubin in theurine. 3.30.8.8free acid. ‘Strong acid’, 11.9frequency. Passing urine very often, 8.5Fuchs-Rosenthal counting chamber (ML 124). A countingchamber O-2 mm deep, 8.1 I, 9.9,13.18fungi. A group of organisms also known as moulds, 8.13,11.15funneZ(ML 25). An instrument tostopliquids spilling whenthey are to be poured into something with a narrowmouth, 2.2,13.8bgallipot. A small pot or basin, 9.4galvanometer. An instrument for measuring electricity,5.16gametocyte. A stage in the life of the malarial parasite,7.33gas. Air is a mixture of gases; a special kind of burnablegas is used in the Bunsen burner, 3.4gasket. A rubber ring or washer, 1.21gastric. Belonging to the stomachgastric juice. The liquid that is made by the stomach todigest food, 11.9gastric washings. A way of obtaining sputum from childrenfor examination, 3.20, 3.21, 11.4agauze. (i) A thin cotton cloth used in hospitals. (ii) Acloth made of metal wires (ML 26), 12.9gel. When a liquid goes nearly solid it is said to form ajelly or gel, 7.40genus. The tribe to which an organism belongs, 1.13Giardia lamblia. A flagellate which causes diarrhoea,10.10giardiasis. The disease caused by Giardia, 10.10giving set. Equipment used to give blood, 12.9glass tube (ML 5 1). 3.9glassware. Equipment made of glassglobulin. One of the plasma proteins (two others arealbumen and fibrinogen), 7.40globus, globi. Many Mycobacterium leprae close togetherinside a cell are said to form a globus, 11.1 Idglucose. The most important sugar in the body, 8.6glycosuria. Sugar in the urine, 8.2gonococcus. Another name for Neisseria gonorrhoeae,11.6GPR. General purpose reagent, 3.15graduated. Divided in a way that can be used to measurethings, 1.3,5.7graduated pipette. A pipette marked to hold knownvolumes of fluid, 5.7graduation marks. The marks or lines used to graduatesomething, 1.3gram. A small weight in the metric system, usually shortenedand written ‘g’

VocabularyIndexGram’s method. A way of staining bacteria, 11.5granules. Small pieces of a solid, 1.4graph. A special ‘picture’ for doing arithmetic, 5.2 lbgrease pencil (ML 3 1). A special pencil with a greasylead that will write on glass, 2.2, 3.11, 13.8bGrey wedge jhotometer (ML 115). A measuring instrumentfor coloured solutions, 2.5, 5.11, 9.13, 13.13gross. Great, very large, very many, ‘very positive’, etc.,4.4gur. This is the whole of the tube that joins the mouth tothe anus. The intestines form part of itHhaemurocrit. The same as the packed cell volume, 7.2haemocytobhzst. The parent cell from which blood cellsare formed, 7.17, Picture A, FIGLXE 7- 10haemoglobk. The red substance inside red blood cells,1.9, 5.9, 1.1, 7.7, 7.24, 8.13haemoglobin diluting&id. A fluid used for measuringthe haemoglobin, 3.3 1, 7.1haemoglobin solubility method. See 7.26haemoglobinopathy. A disease caused by abnormalhaemoglobm, 7.24, 7.25, 7.27haemolyse, haemo!vsk The breaking open and destructionof red blood cells, 1.18, 7.9haemoiytic anaemia. An anaemia due to destruction ofblood inside the body, 7.9, 7.24, 7.32, 8.8Haemophirtrs inpUenzae. One of the bacteria which causemeningitis, 9.16Haldane scale. A way of measuring haemoglobin using ascale which runs from 0 to lOO%, 5.13head injury, CSF in. See 9.19health centre laboratory. A general description, 3.46helminth. A worm, 1.14hepatitis. An inliammation of the liver, 4.8Heterodera radicicola. See 10.6Heterophyes heterophyes. See 10.6‘Hibitane’. An antiseptic, 12.12, 12.13histology. The study of tissues, 4.10histow. A medical history is the story of what has gonewrong with a patient (Second Preface)holder (ML 28). A loopholder, 3.19hollow. Empty, with a hole inside, 1.3holly 1eaJ A leaf with several sharp points; one of theshapes taken up by sickle cells, 7.25hookworm. A small worm that lives in the small gut,10.5hookworm anaemia. Anaemia due to hookworm, 7.6horizontal. Flat, 1.3hyaline. Like glass, 8.13hydrated. Containing water, 2.4hydrochloric acid (ML 94c). A clear caustic liquid usedfor making acid alcohol and Ehrlich’s reagent, 2.4,3.16, 11.1hygroscopic. A hygroscoyic chemical takes water out ofthe air and becomes wet, 2.4Hymenolepis nana. See 10.6hyper--. More or too muchhyperglyaemia. Too much sugar in the blood, 8.6hypertonic. Having a greater salt concentration than thecell cytoplasm, 1.18hype-. Less, too littlehypogZycaemia. Too little sugar in the blood, 8.6hypotonic. Having a smaller salt concentration than thecell cytoplasm, 1.18I-iasis. This ending to a word means an infectious diseasecaused by a worm or a protozoan, 1.12,7.38Imrd. A firm who make filters, 5.18immahrre. Young, not fully grown, 1.3immersion oil (ML 96). A special oil used with the oilimmersion objectives of microscopes, 2.4, 6.7, 6.14,13.10inaccurate. Not exact, rough, 1.3incompatible blood. Blood is incompatible for a patient ifagglutination takes place when it is mixed andincubated with his serum, 12.1incubate. To incubate something is to keep it warm,12.6indicator. A chemical which changes colour when the pHchanges, and in doing so tells us what the pH is,1.7itlfant. A young child, 1.3infec!iq infected. We are said to be infected by a parasitewhen we have it living inside us. We suffer froman infection with that parasite, 1.12infectious disease. Disease due to infection, 1.12infinity. The largest possible number, 1.13injlammable. Very easily burnt, 1.4INH. Isonicotinic acid hydrazide, a drug used to treatpatients with tuberculosis, 2.5, 8.9, 13.19insecticide. A chemical to kill insectsinstrument. A special laboratory machine, 1.3, 2.1insulin. A drug used in treating diabetes, 7.42, 8.6interpupillary distance. The distance between the middle(the pupils) of a man’s eyes, 6.8intestine. The intestine is part of the tube through whichfood passes from the stomach to the anus. It is dividedinto two main parts, the small intestine and the largeintestineintracellular. Inside cells, 11.6intramuscular needle. A needle for giving injections intothe muscles, 9.5,iodine (ML 79). Small dark brown crystals used to makeLugol’s iodine. Also used as an antiseptic, 2.4, 3.32,13.10iris diaphragm. Part of the condenser of a microscopewhich opens and closes to alter the light reaching theobject, 6.3iron. See 7.6iso-. Equal, or the sameisotonic. Having the same salt concentration as the cellcytoplasm, 1.18Jjaundice. The yellowing of a patient’s tissues, 4.8, 8.8,

VocabularyIndexKKahn tube (ML 48~). A middle size of test tube, 2.2Kerrrig’s sign. One of the signs used to diagnose meningitis.9.3knurled. A knurled knob or ring is one which has arough edge so that it can be easily turned and does notslip through your fingers, 5.14kwashiorkor. A form of protein joule malnutrition(PJM), 8.4, 10.12Llabelling bottles, etc. See 3.11‘Labogar’. A kind of gas which is sold in small tins, 3.4,13.30laboratory A medical laboratory is a room with specialequipment (machines) for finding things out aboutpatients (Second Preface)lactase. An enzyme which digests the sugar called lactose,10.12lactose intolerance. The inability to digest and absorblactose, 10.12Leishman’s bufir. A buffer made of phosphate saltsused with Leishman’s stain, 3.20, 7.12Leishman’s stain. A stain used for blood films, 1.7, 3.33,7.12, 7.13leishmmiasis. A disease caused by a protozoan parasitecalled Leishmania donovani, 7.40lens. A piece of smooth curved glass for bending light.Spectacles have lenses, 5.11, 6.2lens tissue (ML 29). Special soft paper for cleaninglenses, 2.2, 6.16, 13.8bleprosy. A chronic disease due to infection withMycobacterium leprae, 1.12, Il. 1, 11.1 Ilesion. A diseased place in the body, I 1.11leucocyte. A white blood cell, 7.14leucocytosis. An increased number of white cells in theblood, 7.21, 7.29leucopenia. Too few white cells in the blood, 7.2 1leukaemias, myeloid and lymphatic. A rare and seriousblood disease in which there are too many white cellsin the blood, 7.21It@ cycle. The circle of stages through which an organismpasses during its life, 7.32liner. The black rubber circle inside the cap of a universalcontainer or bijou bottle, 2.2liquid. Something which flows and takes on the shape ofthe bottle or cup it is in, 1.4Lou loa. A filarial worm, 7.37local anaesthetic. A drug such as procaine which isinjected into the tissues to stop the patient feeling painin that area, 9.5, 12.12loop. A loop is a ring or circle of wire or string. A wireloop is used as an easily sterilized spoon for smallquantities of specimens, 3.5, 3.10, FIGURE 3-7loop-holder (ML 28). A metal ‘pencil’ with a chuck atone end for holding wire, 2.2, 3.10, 13.8bLovibond cell (ML 48d!. A special square tube which isonly used with the Lovihond comparator, 2.2, 5.10,Picture C, FIGURE 5-5Lovibond comparator (ML 13). A machine for comparinga test solution with coloured glass standards, 2.5,---5.10, 7.1, 7.41, 7.42, 13.8bLovibond discs (ML 2 1). Black plastic wheels withseveral coloured glass standards around their edges.There are discs for haemoglobin, blood sugar, andblood urea, 2.2, 5.10Luerfitting. One of the sizes of fittings for syringes andneedles, 12.9Lugol’s iodine. An iodine solution used for Gram’smethod and for staining protozoa in the stool, 3.32,11.5, 10.2lumbar puncture. Putting a needle into the lower part ofthe arachnoid space to obtain CSF, 9.1, 9.2, 9.4,9.5lymph. A thin fluid that comes out of the capillaries andis taken back to the blood through the lymph vessels,7.37&mph nodes. Bean shaped organs in the groins, and inmany other parts of the body where lymph is filteredand in which lymphocytes are made, 11. I2lymph vessels or lymphatics. Small thin tubes which takea fluid called lymph back to the blood, 7.37lymphocyte. A kind of white blood cell, 7.14, 7.16,Pictures A and C, FIGURE 7-9&se. To burst and break open. To dissolve, 1.18lysol. A common disinfectant, 1.19, 2.3, 3.12, 13.8aMmacrocyte, macrocytic. In a macrocytic blood film manyof the red cells are larger than normal, 7.19macrocytic anaemia. A t-jrpe of anaemia in which abnormallylarge red r;el& are seen in the blood, 7.19macrophage. A ceil which is able to ‘eat’ bacteria andother small particles, 10.7magnification. The number of times bigger an object ismade to look, 6.7magn@ngpower. The power of an objective or eyepieceto make an object look bigger, 6.7malachite green (ML 107). A green stain used for counterstainingin the Ziehl-Neelsen method, 2.4, 3.34,Il.1malaria. A disease caused by protozoan parasites of thegenus Plasmodium, 7.5, 7.9, 7.32, 7.33, 8.8male. Man or belonging to a manMansonella ozzardi. A filaria! worm, 7.37mantle. The cloth part of a paraffin pressure lamp thatgets white hot, 6.11manual. Need for standard manual, 13.1, 13.3 1marrow. Red marrow is a tissue inside bones in whichblood cells are formed, 7.17mask. A surgeon’s mask is a piece of loose cloth whichgoes over his nose and mouth. It catches any drops offluid that may contam micro-organisms, 9.5match. Something is said to match something else whenit is equal to it in weight or colour, etc., 5.9mature. Fully grown. Rdult, 1.3MCHC. The mean corpuscular haemoglobin concentration,7.3, 7.19

Vocabularyindexmeasuring qiinder (ML 1 S and 19). A tall glass jar formeasuring liquids, 2.2,5.7meat$bres. Partly digested meat seen in the stools, 10.5mechanical stage. A machine which moves a slide on thestage of a microscope, 6.10megakap&tes. Large cells in the marrow from whichplatelets are formed, 7.15megatoblust. An abnormal type of immature red cell,7.19metaena stool. A stool which is black because it containsmuch partly digested blood, 10.11membrane. A very thin covering, 1.3meninges. The covering of the brain, 9.1meningitis. Inflammation of the meninges of the brain,9.1, 9.3, 9.16,9.17,9.18meningoc Another name for Neisseria meningitidis,l-23,9.16meniscus. The curved surface of a liquid, 5.7menorrhagia. Very heavy monthly periods, 7.6mercury. A heavy metal which is liquid at the temperatureof a room, 2.2merozoite. A stage in the growth of the malarial parasite,2.32Wetagonimus yokogawai. See 10.6metamyelo~~~fe. An immature white blood cell, 7.17,Picture E, FIGURES 7-9 and 7-10methbnol or methyl alcohol (ML 99). This is a light,volatile, inflammable liquid used for makingL&&man’s stain, 2.4,3.33, 7.12methylene blue (ML 108). Deep blue crystals used as acounterstain in the Ziehl-Neelsen cold method, 2.4,3.35, 11.1methytene in acid alcohol. A solution used in the coldZiehl-Neelsen method, 3.35, 11.1metric system. The way of weighing and measuring usingmillilitres, grams, and centimetres, 5.1mg A milligram or a thousandth part of a gram, 5.2mg %. The number of milligrams of a substance thatthere are in 100 ml of a patient’s plasma, 5.8microcytic. In a micro&c blood film many of the redcells are too small; a microcyte is a small red cell, 7.6,7.19microfilariae. Larval (young) filarial worms, 7.37, Il. 14microhaematocrit. A way of measuring the haematocritwith a very small volume of blood, 7.2micron. This is one thousandth of a millimetre or a millionthpart of a metre, it is written ‘{lrn’, 6.1micro-organism. Very small organisms, 1.11microscope (ML 30). An expensive machine for lookingat things which are so small that they cannot be seenby the eye alone (Chapter 6)millilitre. One thousandth part of a litre, written ‘ml’. 5.1,5.7mirror of a microscope. See 6.3ml A millilitre or a thousandth part of a litre, 5.1,5.7mobile. Moving, 3.7, 8.14Mohr’s clip. A special instrument for clipping or closinga rubber tube, 3.3monocular. One-eyed, a microscope with one eyepiece,6.8monocyte. A kind of white blood cell, 7.14, Picture B,FIGURE 7-9motile. Able to move, 1.14, 8.14, 10.7motility. Movement, 8.14, 10.7mouthpiece (ML 33). A short piece of smooth glass tubeattached to a rubber tube: used for filling pipettes bysucking through, 7.1, 13.&bMRC blood transfusion equipment. Equipment designedby the Medical Research Council of Great Britain,12.9mucosa. The lining or inner surface of an organ like thegut or the bladder, 10.8mucus. A sticky white substance made by some epithelialcells, 10.1mycelia. The long hair-like branching cells of’a mould orfungus, 8.13, 11.15mycobacteria. The genus of bacteria causing tuberculosisand leprosy, 11.1Mycobacterium leprae. The parasitic micro-organismcausingleprosy, 1.12, 11.1, 11.11Mycobacterium tuberculosis. The parasitic microorganismcausing tuberculosis, 1.12, 11.1myeloblast. A kind of very young white cell which willgrow to become a polymorph, 7.17, Picture B,FIGURE 7- 10myelocyte. An immature white cell, Picture D, FIGURE7-10Nnasal. From the nose, 11.1 lcNecator americanus. One of the hookworms, 10.5‘Needle and tube’. A way of taking blood using only alarge needle and a piece of rubber tube, 4.9negative. Absent or not there, 1.3Neisseria gonorrhoeae or the gonococcus. The cause of avenereal disease called gonorrhoea, 11.6Neisseria meningitidis or the meningococcus. One of thebacteria which often cause meningitis, 9.16nematode. A round worm, 1.14nephritis. A kidney disease, 8.4nephrotic syndrome. A kidney disease, 8.4Neubauer counting chamber (ML 15). A special kindof counting chamber 0.1 mm deep, 7.29, 9.9,13.8bneutral. Neither acid nor alkaline, but between themboth, 1.6neutrophil polymorph. The common kind of polymorphcontaining many small purple granules. Neutrophilmeans neutral staining, 7.14, Picture G, FIGURES 7-9and 7-10nickel-chrome wire (ML 55). Special strong wire thatdoes not get burnt in a flame, 3.10, 13.8bnodule. A small lump, 11. I 1nomogram. A special figure for doing arithmetic, 7.3non-. Notnormal. If something is seen in healthy people, we say itis normal, 1.3

VocabularyIndexnormobtast. A young red cell with a nucleus, 7.14,Pictures I, J, K, FIGURE 7- 10normochtomic. Normally coloured. A normochromicred cell is one which is normally filled with haemoglobin,7.19notch. A piece cut out of something, 5.2notice-board. Instructions for the ward notice-board, 4.6nozzle. The place on a syringe where the needle fits, 1.2 1nuclear membtane. The ‘coat’ of the nucleus, 1.9.nuclei. More than one nucleusnucteo6 A *hole’ in the nucleus of some immature bloodcells, 7.17nucleus. A large ‘ball’ in the middle of a cell, 1.9nut. Something which turns and fits on to a screw0object. The thing we look at with a microscope, 6.2objective. The part of a microscope which ‘lqoks at’ theobject and sends light up the tube to the eyepiece, 6.2,6.7obZiQue. Sloping. From one sideocclusive. Blocking, 7.27aoccult. Hiddenoccult blood. Blood in the stool that can only be found bydoing a special test, 10.11occult blood reugent. A mixture of barium peroxide andortho-tolidine used to test the stools for occult blood,3.36, 10.11oedema, oedematous. Fluid in the tissues causing them toswell, 8.4Ohaus balance (ML 1). A laboratory balance, 5.1,13.8boil immersion objective. An objective which can onlylook at an object through oil, 6.7oitstone (ML 63). A special stone used for sharpeningtools, 12.10, 13.9Olympus microscope (ML 30). The name of a Japanesefirm who make microscopes, 6.1, 13.8bOnchocerca votvutus. A nematode worm which lives inthe skin, 7.37, 11.14onchocerciasis. The disease caused by the worm calledOnchocerca volvulus, 11.14opaque. Not easily seen through, 1.3operculum. The ‘door’ or weak part in ari ovum throughwhich a larva can leave it, 10.6organ. A part of the body, such as the brain, the heart, orthe spleen, 1.9organism. Any livi;?g thing, 1.11ortho-totidine (ML 80). A chemical used in testing thestools for occult blood, 2.4, 3.36, 10.11-ose. Sugars end in ‘ose’, lactose and glucose for example,8.3, 10.12oval. Egg shapedovum, ova. An egg, or eggs, 1.12, 10.5, 10.6oxyhaemogtobin. A kind of haemoglobin which is redbecause it has combined with (is joined to) oxygenfrom the air. Haemoglobin which is not combinedwith oxygen is purple and is called reduced haemoglobin,7.1Ppacked cell volume, or PCV. The percentage volume occupiedby the packed cells in a specimen of blood, 7.2packed cells. The red cells at the bottom of a tube ofblood which have been prevented from clotting andthen centrifuged hard, 7.2paging numerator. A page numberer or a machine forstamping numbers, 4.3, 12.13Pandy’s reagent and method. A saturated solution ofphenol in water used as a simple test for an abnormalCSF protein, 3.37 (reagent), 9.10, 9.13 (method)pans. The ‘plates’ for holding things on a balance,FIGURE 5- 1para-dimethyl-amino-benzaldehyde (ML 82). A yellowchemical used for making Ehrlich’s reagent, 2.4, 3.27,8.8Paragonimus westermani. See 10.6, 11.4bparasite. An organism which lives on or inside a largerorganism, 1.12parasitology. The study of parasites: often it only meansthe study of worms and protozoa, 4.10parfocal. Objectives are said to be parfocal when they aremounted (held) in the nosepiece in such a way that anobject in focus with one objective will be in focus withall the others, 6.7particle. A very small piece of a solid, 1.4parts’. Measures, 5.8PAS. Para-aminosalicylic acid, a drug used for treatingpatients with tuberculosis, 3.38, 8.9PAS test strips. Strips of paper soaked in ferric chlorideused to test the urine for PAS, 3.38, 8.9Pasteur pipette. A special pipette made in the laboratoryfrom glass tubing and used with a rubber teat, 3.9PCV. See packed cell volume or haematocrit, 7.2pellets. Small balls, 2.4penis. A man’s sex organ, 11.6percentage or ‘%‘. The number of something in everyhundred of something else, 5.8‘Perspex’. A light, clear plastic like glass, 1.3, 7.2PH. A way of measuring acidity and alkalinity, 1.7pH of the stools. See 10.12phenol (ML 81). A caustic, oily liquid when hot or crystalswhen cold: also called carbolic acid. Used forPandy’s reagent, 2.4, 3.37, 9.10, and carbol fuchsin3.22b, 3.23phosphates. See 2.4, 3.20physiological saline. Isotonic saline, 1.18pia mater. The soft inner covering of the brain, 9.1pigment. A coloured substance, 7.32pilot bottle. A small bottle of blood fixed to the mainbottle of a donor’s blood from which specimens fortesting are taken, 12.11pipette (ML 32, 35). A glass tube for holding or measuringvolumes of liquid. See also Pasteur pipette andgraduated pipette, 2.2, 5.7, 7.1, 13.8bplane. Flat, 6.3plasma. The liquid part of blood, 1.5, 1.17plasmodium. The name of the genus (tribe) to whichmalaria parasites belong, 7.32, 7.33

Vocabulary IndexPlasmodium, fatciparum, vivas, ovate, and matariae. Thefour species of malaria parasite, FIGURE 7-29plastic. This often means soft and easily bent. Used hereit means a group of substances from which manyuseful things are made, 1.3ptasricine (ML 67). A kind of mud or clay which doesnot get dry, 2.3, 3.2, 3.11platelets. Small pieces of cells found in the blood, 7.15,Picture H, FIGURE 7-9pliers (ML 68). A tool for cutting and holding things,Picture 4, FIGURE 7-6plug. A plug is something which can be pushed into ahole, 3.6plugged. Blocked or filled upplunger. The inside part of a syringe, FIGURE 4-3plus notation. A special way of writing reports, 4.4pne2mococcus. Another name for Streptococcuspneumoniae, 9.16poikitocy~osis. Abnormally shaped red cells, 7.19pointer. A hand which points to the graduations on thescale of a balance or galvanomerer, 5.2, 5.16poise. To poise something is to balance it, 5.2poising nut. The nut on a balance which makes it swingevenly, FIGURE 5- 1polychromasia. Many polychromatic red cells in theblood, 7.19, 7.23potychromatic. A polychromatic red cell is a young redcell stained with Leishman’s stain It looks purple,7.19, 7.23, Picture D, FIGURE 7- 11poijmorph. A polymorphonuclear leucocyte, 7.14potymorph Ieucocytosis. An abnormally high number ofpolymorphs in the blood, 7.21‘Polypor (ML 14~). A special plastic container forsputum, stools, etc., 2.2, 3.12,4.6polypropylene. A special strong kind of plastic that canbe autoclaved without spoiling, 1.3‘Potystop bottle’ (ML 7). A special bottle with a polythenestopper, a pipette, and a teat, 2.2, 7.11, 7.12, 13.8bpotyrhene. A common kind of plastic, 1.3‘PoZylube’ (ML 14d). A special plastic container forblood, etc., 2.2, 3.12, 4.6positive. Present, 1.3potassium cyanide (ML 125). A very poisonous chemicalused to test for INH in the urine, 5.7, 8.9, 13.19porassiumfluoride (ML 84). A white powder used to stopthe cells of the blood or CSF breaking down (eating)sugar, 2.4, 4.6, 7.42, 9.15potassium iodide (ML 85a). White crystals used in makingLugol’s iodine, 2.4, 3.32powder. Something made of small pieces of a solid: sugaror salt for example, 1.4precipitate. Solid particles formed in a liquid, 1.4pregnunt. A woman is said to be pregnant when there is achild inside herpreset focus lock. A lock on the Olympus microscopewhich lets us focus on a second slide without having touse the coarse adjustment, 6.12pressure cooker (ML 69). A small autoclave, 1.20, 1.21,9.4pressure stove (ML 71). A stove, such as a ‘Primus’ stovewhich uses paraffin or kerosine under pressure, 1.20,3.9Primaquina A drug used in treating malaria, 7.33prism. A glass block for bending light, 5.11, 6.2, 6.9proctoscope. An instrument for looking through the anusat the last 10 cm of the gut, 11.13productive cough. A cough in which sputum is produced,11.4a‘Progressive directional crawl’. The way EntamoebahistotJtrica moves, 10.7prom-vetocyte. A young white cell which will grow tobecome a polymorph, 7.17, Picture C, FIGURE 7- 10protein de$ciency anaemia. Anaemia due to lack of proteinin the food, 2.8proteinometer (ML 36). An instrument for measuringprotein in the CSF, 2.2,9.13prooteins. Complicated substances from which cells aremade, 1.10, 7.8proteinuria. Protein in the urine, 8.2, 8.3protozoan, protozoa. A single celled micro-organismwith a nucleus, 1.14, 10.7pseudopodium, pseudopodia. The ‘foot’ of an amoeba,1.14, 10.7pulp. Soft tissue, 11.11puncture. Putting an instrument into something, or makinga hole in it, 9.2, 11.12pupils. The dark middle parts of the eyes, 6.8purple. A colour made by mixing red and bluepurulenf. Like pus, 7.14, 8.1, 8.4, 8.11pus. A thick, usually yellowish liquid made of millions ofdying polymorphs. A pus cell is a polymorph, 7.14,8.1, 8.4, 8.11pus cells in urine. See 8.4, 8.11putrefy. To rot or go bad, 1.15pyuriu. Pus in the urine, 8.4, 8.11Rrack. Something for holding things, such as the test tuberacks, ML 40 and 4 1, the slide rack, 3.11, or thestaining rack, 3.2rainbow. A curved line of colours seen when sun shineson rain, 5.12reagent. The chemicals used in a ‘Method’. Many reagentsare solutions of chemicals in water, 3.15 to 3.45reagent, prepared centrally. See 13.11, 13.25recipient. A patient to whom blood is given, 12.1record. Knowledge that is written down so that it is notlost, 4.1Record @ring. One size of fittings for syringes andneedles, 12.10rectal. Belonging to the rectum which is the last part ofthe gut before the anus, 11.13red blood cell. See 1.9, 7.19reducing valve. A tap which lets the high pressure gas outof a cylinder slowly and at low pressure, Picture F,FIGURE 3-2, 3.4refrigerator. A special box with a machine that keeps itsinside cold, 12.3, 12.14

,Vocabulary IndexPlasmodium, falciporum, vivay? ovate. and malariae. Thefour species of malaria parasite, FIGURE 7-29plastic. This often means soft and easily bent. Used hereit means a group of substances from which manyuseful things are made, 1.3plasticine (ML 67). A kind of mud or clay which doesnot get dry, 2.3,3.2.3.11platelets. Small pieces of cells found in the blood, 7.15,Picture H, FIGURE 7-9priers (ML 68). A tool for cutting and holding things,Picture 4, FIGURE 7-6plug. A plug is something which can be pushed into ahole, 3.6plugged. Blocked or filled upplunger. The inside part of a syringe, FIGURE 4-3pIus notation. A special way of writing reports, 4.4pne2mococcus. Another name for Streptococcuspneumoniae, 9.16poikikxytosis. Abnormally shaped red cells, 7.19pointer. A hand which points to the graduations on thescale of a balance or galvanometer, 5.2, 5.16poise. To poise something is to balance it, 5.2poising nut. The nut on a balance which makes it swingevenly, FIGURE 5- 1polychromasia. Many polychromatic red cells in theblood, 7.19, 7.23polychromatic. A polychromatic red cell is a young redcell stained with Leishman’s stain. It looks purple,7.19, 7.23, Picture D, FIGURE 7-11poljmorph. A polymorphonuclear leucocytel 7.14polymorph leucocyrosis. An abnormally high number ofpolymorphs in the blood, 7.21‘Polypot’ (ML 14~). A special plastic container forsputum, stools, etc., 2.2, 3.12,4.6polypropylene. A special strong kind of plastic that canbe autoclaved without spoiling, 1.3‘Polystop bottle’ (ML 7). A special bottle with a polythenestopper, a pipette, and a teat, 2.2, 7.11, 7.12, 13.8bpolythene. A common kind of plastic, 1.3‘PO/y&be (ML 14d). A special plastic container forblood, etc., 2.2, 3.12, 4.6positive. Present, 1.3potassium cyanide (ML 125). A very poisonous chemicalused to test for INH in the urine, 5.7, 8.9, 13.19potassiumfluoride (ML 84). A white powder used to stopthe cells of the blood or CSF breaking down (eating)sugar, 2.4, 4.6, 7.42,9.15potassium iodide (ML 85a). White crystals used in makingLugol’s iodine, 2.4, 3.32powder. Something made of small pieces of a solid: sugaror salt for example, 1.4precipitate. Solid particles formed in a liquid, 1.4pregnunt. A woman is said to be pregnant when there is achild inside herpreset focus lock. A lock on the Olympus microscopewhich lets us focus on a second slide without having touse the coarse adjustment, 6.12pressure cooker (ML 69). A small autoclave, 1.20, 1.21,9.4pressure stove (ML 71). A stove, such as a ‘Primus’ stovewhich uses par&n or kerosine under pressure, 1.20,3.9Primaquine. A drug used in treating malaria, 7.33prism. A glass block for bending light, 5.11, 6.2, 6.9proctoscope. An instrument for looking through the anusatthelast lOcmofthegut, 11.13productive cough. A cough in which sputum is produced,11.4a‘Progressive directional crawl’. The way EntamoebahisroZy,tica moves, 10.7promyelocyte. A young white cell which will grow tobecome a polymorph, 7.17, Picture C, FIGURE 7- 10protein deficiency anaemia. Anaemia due to lack of proteinin the food, 2.8proreinometer (ML 36). An instrument for measuringprotein in the CSF, 2.2,9.13proteins. Complicated substances from which cells aremade, 1.10, 7.8proteinuria. Protein in the urine, 8.2, 8.3protozoon, protozoa. A single celled micro-organismwith a nucleus, 1.14, 10.7pseudopodium, pseudopodia. The ‘foot’ of an amoeba,1.14, 10.7pulp. Soft tissue, 11.11puncture. Putting an instrument into something, or makinga hole in it, 9.2, 11.12pupils. The dark middle parts of the eyes, 6.8purple. A colour made by mixing red and blueputulent. Like pus, 7.14, 8.1, 8.4, 8.11pus. A thick, usually yellowish liquid made of millions ofdying polymorphs. A pus cell is a polymorph, 7.14,8.1, 8.4, 8.11pus cells in urine. See 8.4, 8.11putrefy. To rot or go bad, 1.15pyuriu. Pus in the urine, 8.4, 8.11Rrack. Something for holding things, such as the test tuberacks, ML 40 and 41, the slide rack, 3.11, or thestaining rack, 3.2rainbow, A curved line of colours seen when sun shineson rain, 5.12reagent. The chemicals used in a ‘Method’. Many reagentsare solutions crf chemicals in water, 3.15 to 3.45reagent, prepared centrally. See 13.11, 13.25recipient. A patient to whom blood is given, 12.1record. Knowledge that is written down so that it is notlost, 4.1Record fitting. One size of fittings for syringes andneedles, 12.10rectal. Belonging to the rectum which is the last part ofthe gut before the anus, 11.13red blood cell. See 1.9, 7.19reducing valve. A tap which lets the high pressure gas outof a cylinder slowly and at low pressure, Picture F,FIGURE 3-2, 3.4refrigerator. A special box with a machine that keeps itsinside cold, 12.3, 12.14

VocabularyIndexrelapse. When a patient gets well for a time and then getsill again he is said to relapse, 7.35relapsing fayer. An illness caused by Borrelia duttoni,7.3 1, 7.35, Picture E, FIGURE 7- 11report. Something found out about a patient which is sentfrom the laboratory to the wards, 4.1request slip. A small piece of paper sent to the laboratoryasking for a method to be done on a patient, 4.1reticulo~vte. A young red cell stained with brilliant cresylblue. It looks like a blue ‘net’, 7.17, 7.23, 7.28reticuloc~tosis. Many reticulocytes in the blood, 7.17,7.23, 7.28retract. To get smaller, 1.17revolving nosepiece. The part of a microscope whichholds the objectives, 6.7Rhesus group. One of the systems (kinds) of bloodgroups, 12.1, 12.7rigor. When a patient has a rigor he feels very cold, heshakes all over and his temperature rises, 12.9rod. Something long and thin likcKa pencil, 1.3Rothera’s reagent. A powder used for testing the urinefor acetone: there are two kinds of Rothera’s reagent,3.39y8.7rouleaux. Red cells piled on top of one another like a pileof coins. 7.13. 12.6. Picture F, FIGURE 7- 11routine. The way in which things are done day by day,3.13ruled area. Some very small squares drawn on a countingchamber, 7.29Ssafe@ plug. Part of a pressure cooker which lets thesteam out before the pressure inside the cooker getstoo high and bursts it, 1.21saline. A solution of salt in water, 1.4, 1.18, 3.40saline stool smear. See 18.2asalt. See 1.6saturated. A saturated solution of a substance is onewhich will not dissolve any more of that substance, 1.4saturated sodium acetate solution. A reagent used inEhrlich’s test for urobilinogen, 3.4 1, 8.8s&e. (i) A row of lines used to measure; a ruler hasseveral scales, 5.2. (ii) The size something is drawn ina picture, 1.1scalpel. A surgeon’s knife, 1.20, 11.15Schistosoma haematobium. A worm which lives in theveins of the bladder, 7.6, 8.13, 8.15, 10.3Schistosoma mansoni. A worm which lives in the veinsin the wall of the lower part of the gut, 7.2, 10.3Schistosoma japonicum. See 10.6schistosomiasis. Bilharziasis or infection with schistosomeworms, 8.4, 8.15schizont. A stage in the life cycle of the malarial parasite,7.32, 7.33Schu&%er’s dots. Dots seen in the red cells when they areinfected by some species of malarial parasite, 7.34scope. Words ending in scope are all instruments forlooking at something, 6.1 (microscope), 11.13(proctoscope)scrapings. Small pieces that have been scraped or rubbedoff something, 11.15scum. A ‘skin’ on the top of a liquid, 7.12 -seal. To seal something is to close or cork it, 7.23, 7.25,10.1segment. A part of something, 7.14, Picture G, FIGURE7-10selenium. A substance which makes electricity whenlight falls on it, 5.16, 5.25‘Sellotape swab’. A way of finding the ova of Enterobiusvermicularis, 10.4semen, seminaljluid. A man’s seed, 11.10sequestrene (ML 83). A chemical which stops bloodclotting, 1.17, 2.4serology. The study of sera, 4.10serum, sera. The yellow liquid that comes out of bloodwhen it clots, 1.17shaft. A rod which turns, 1.5 : the tube of a needle, 12.12sheath. A covering which protects whatever is inside it,2.3, 7.37shelves. See 3.1, 3.46shutters. Things which alter the amount of light gettingthrough them, 5.16sickle-cell, anaemia, crisis, disease, trait. See 7.24, 7.25,7.26, 7.27sigmoidoscope. An instrument for looking through theanus at the last 25 cm of the gut (the sigmoid colon),11.13signs. Something that is observed to be wrong with apatient, such as a pale tongue, a swelling, or a sore, 1.3silicone tubing. A special kind of ‘rubber’ tubing made ofa substance called silicone, 12.9sink. A sink is a kind of basin with a tap, 3.2siphon. A way of making water run upwards before itruns a longer way downwards, 3.3skin scrapings for fungi. See 11.15slide (ML 37). A piece of glass on which things are putwhen they are looked at with a microscope, 6.2slide rack. Something for holding slides while they dry,3.11slip. A small piece of paper, 4.1slit. A narrow space, 5.16slot. A long hole, 6.16smear. A specimen spread thinly on a slide, 7.11, 10.2,11.11snip. A small piece that has been cut off, 11.14socket. The hole into which a plug fits, 3.7sodium acetate (ML 86). White crystals used in Ehrlich’stest, 2.4, 3.27, 8.8sodium azide (ML 128). A very poisonous chemical usedto preserve sera, 2.5,4.10sodium carbonate (ML 87). A white powder used inmaking Benedict’s reagent, 2.14, 3.16,8.3, and in testingthe urine for acetone, 3.39, 8.7sodium chloride (ML 88). ‘Common salt’, a white powderused for making saline, 1.4, 1.18sodium citrate (ML 90). A white powder used in solutionas an anticoagulant in the Westergren ESR, 2.4,3.42a, 7.40

Vocabulary index‘Odti” dithion’re (h’tL 93b). A chemical used to test the stool. The waste from the bowel or gut, Chapter TenbI& for sick@a 2 Q 7.25, 7.26stool smear. See 10.2a, 10.2bso#unt hj?droxide & i il). Hygroscopic white pellets stopper. A c&p or cork, 2.2vhich are dissolv W to make a strong solution that is streaming. A kind of movement, 8.14usd for looking %t skk scrapings for fungi, 11.15 streptococcus. A COCCUS growing in chains like a string of‘Ofid* something Whi& does not flow and has its own beads, 9.16shape, 1.4Streptococcus.pneumoniae. One of the bacteria whichsolution. A liquid in \vhich something is dissolved, 1.4 often cause meningitis, 9.16spares. parts Of YQhr equipment YOU should keep to streptomycin. A drug used for treating tuberculosis, 11.4feNace Parts *at break, 2.6,5.22Strongyloides stercoralis. A worm living in the gut, 10.6SpahllU (ML 38). A Vhemist’s spoon, 2.2stud. A short rodspecies- The name 0F a @c&u organism, 1.13stylet. The wire that goes down inside a needle, 9.4‘ficim% part Of ‘Qme&ing that is to be looked at. A substance. Anything which is the same all through, 1.4spcrmen is part Qf a p&t’s stool, urine, blood, etc. sugar in blood. See 7.42(Second Preface), Chapter Four, 8.1&phones. Drugs used in treating leprosy, 3.43a, 8.10‘fictnrm, The ““% which make up white light; red, sulphosalicylic acid (ML 73). A white powder used forOrage ye”owT gpQn blue ti01d, 5.12testing the urine and the CSF for protein, 2.4, 3.43b,sPem’*m- An inst%&t to help you to see something 3.44, 8.3,9.13more easily, 11.1 qcsulphuric acid (ML 95). A thick, clear, caustic,spermazozo~ The ce hs h the seminal fluid, 11.10DANGEROUS liquid used for making dichromates.emS- Short for ~~~~~~~ 11.10cleaning fluid; dilute sulphuric acid is also used tosph~gntomanometel. A machine for measuring the pres- measure the blood sugar, 2.4, 3.12,3.26Ore Of blOOd in 6, ar&ries, 12.12supernatantfluid. The fluid on top of a deposit, 1.4spirt. TO Nm round b cry fast, to centrifuge, 1.5suppurative meningitis. Meningitis in which there is pusspifil(ML “*)’ A %aufe, mostly ethyl alcohol. It is a in the meninges, 9.16fuel for the spirit $ amp, a mild antiseptic, and is also suspension. Particles (such as blood cells) hanging in ausd for m*ing %&)I fuchsin, 2.4, 3.22liquid (such as plasma) form a suspension, 1.4Spirit lamp (ML 39>, A lamp for heating which bums swab. A piece of gauze or cotton wool used for soakingspirit, 2.2up a fluid. To swab something is to soak up the fluid‘priader A ‘lJecial ‘$de for spreading blood films, 7.11, on it. To swab part of the body with antiseptic meansI. 12to paint or cover it with antiseptic, 1.22spring-loaded. A sps g-loaded objective is one which swollen. Large, fat, bigsprings back whes it touches a slide, 6.7symptom, Something that the ,patient says is wrong withsped- A very small sbde I 1.1 lchim, such as a pain, or feeling sick, 1.3spWm’ what a patie%l, &ughs or spits up, 11.1syringe. See 3.12,4.9sputum-)legafive. No WB present in the sputum, 11.1 syringejaundice. A very bad kind of jaundice that can bespaturn-positive. e I3 present in the sputum, 11.1given to a patient by giving him an injection with astsge. 7he part of a 8. lcroscope on which slides are put, dirty needle, 4.86.9StQifl. A coloured cb emical used to colour specimensTbefofe *ey are ‘%&A at with a microscope, 1.16, Taenia solium and Taenia saginata. Tapeworms, 10.1,1. 1510.2, 10.5s&ing rack for slid& S6 P3’)taking blood. See 12.12Stand (a 40, 41, oh 42):‘&m&in Ig for holding or taking set. Equipment used to take blood, 12.9kWing things in. %e same as a rack, 2.2tare. Something to balance the weight of the container instandard. Something whose weight, depth or colour or which it is being weighed, 5.2vo1ume7 etc’7 we ab~ sure about, 5.9, 5.10, 5.11, 5.19 target cell. A red blood cell with a thickening in thestandard block. A pi hce of wood which comes with the middle which is seen in some blood diseases,Grey wedge and %ich contains a grey glass standard, especially sickle-cell anaemia, 7.19, 7.27a5,14TCE. A shortened way of writing ‘TetraChlorEthylene’,sreetorrhoea. Diarrhs l&‘ul .:*I. ,,IU11a mm-h nnrliwctcd ..-u.b’“‘v- A... fat in .I. thP U.” 7.6spols, 10.10teat (ML 43). A little rubber bag which fits a Pasteur-“, 6umYY’Y, a. “V, d. 1”pipette, 2.2, 3.9all the micro-organisms in ‘TeepoP (ML 98). This thick pale yellow liquid is a commonlyused detergent, 1.3, 2.4, 11.1terminals. Screws on an instrument to take electricity toit or from it, 5.16s&kg rod* A rod fo* stirring liquids, 3.9test solution. A solution whose depth of colour we wantStock. A stock of the%. IC& is the supply you keep, 2.6 to measure, 5.9, 5.10

VocabularyIndextest rube (ML 48). A short thick glass tube in which testsare done, 2.2, 13.8btetrachlorethylene. A drug used to treat hookworm infection,7.6thermometer (ML 3 I). An instrument for measuring temperature,2.2, 12.6thick bloodfilm. See 7.3 1thick stool smear. See 10.21?thin bloodfilm. See 7.1 ,Ithrombocytopeniu. Too few platelets (thrombocytes) inthe blood, 7.15tile (ML 49). A flat square of plastic with $oles in it, inwhich some tests are done, 2.2, 8.9time and motion study. The study of the way of doing themost work with the least time and effort, 3.14tissue. (i) The different parts from which the body ismade, such as liver tissue, muscle tissue, skin tissue,etc., 1.9. (ii) Thin paper, such as lens tissue (ML 29),6.16toxuemia of pregnancy. A disease some mothers getwhen they are pregnant, 8.4trachea. The tube which takes air down the neck to thelungs, 1.9transfusion. Taking blood from one person and giving itto another person, 12.1transfusion reaction. The illness a patient suffers from ifhe is given the wrong blood, 12.2transparent. Clear, easily seen through, 1.3trematode. A flatworm or fluke, 1.14trichlorucetic acid (ML 74). Colourless crystals used tomake Fouchet’s reagent, 3.30Trichomonus hominis. A harmless flagellate which livesin the gut, 10.10Trichomonus vaginalis. A flagellate often found in thevagina, 8.13, 11.8Trichuris trichiuru. The whipworm, 10.1, 10.5triple. III three parts.triple beam balance. A balance with three beams (ML l),2.2,s. 1tripod (ML 42). A stand with three legs, 2.2, 7.42trivet. The shelf in a pressure cooker to hold things thatare being sterilized, and thus keeps them out of thewater, 1.21trophozoite. The larger active form of a protozoon, 1.14trunion. The parts of a centrifuge from which the bucketshang, 1.5Trypanosoma gambiense and rhodesiense. Two flagellates,7.36. 9.14. 11.12trFpan&omi&is. The disease caused by trypanosomes,7.30,9.14, 11.12tube of u microscope. An empty tube at the bottom ofwhich are the objectives and at the top of which is theeyepie?e, 6.2, 6.8, 6.9tuberculosis. A chronic disease, usually of the luugs, dueto infriction with Mycobacterium tuberculosis, 1.12,11.1 to 11.4tuberculous meningitis. MeningitisMycobacterium tuberculosis, 9.18causedtubing (ML 50 and 5 1). Long pieces of tube that can bebyused for many purposes, 2.2, 3.4, 3.9turbid. A fluid is turbid when it is cloudy or milky or ifthere are particles floating in it, 1.3, 9.10typical. Usual, ordinary, 1.3Uumbilicus. The ‘hole in +he middle of the abdomen, 9.5undulating membrane. Part of a trypanosome, 7.36universal. Evtiyone, everything, everywhereuniversal container (ML 14a). A specimen bottle with ascrew cap, 2.2,4.6, 13.8buniversal donor. A person of blood Group 0 whoseblood can be given to anyone, 12.2universal indicator test paper (ML 109). A papercoloured with a mixture of indicators which showsmany colour changes and thus many different pH’s,1.8, 10.12universal recipient. A person oi blood group AB who canbe given blood from anyone, 12.2uraemic. Too much urea in the blood, 7.41urea. A waste substance excreted in the urine, 7.41, 8.2ureuse. An enzyme which breaks down urea, 1.10, 7.4 1urethra. The tube taking urine from the bladder outsidethe body, 8.4urethral discharge. A discharge from the urethra, 8.4,11.6urine, clean specimen oJ: See 8.1urinary tract. The parts of the body where the urine is,the kidneys, ureters, bladder, and urethra, 8.1urobilinogen. One of the bile pigments, 8.8uterus. The womb, 7.7Vvucuum. A space with nothing in it, not even air, 4.10,12.9vagina. A woman’s birth passage, 8.4vein. A thin-walled blood vessel taking blood from thetissues to the heart, 4.7venereal. Spread through sex, 11.6venous blood. Blood taken out of veins, 4.7vent. A small hole to let something out. 1.2 1vertebrae. The bones of the spine, 9.1vertebral column. The spine or backbone, 9.1vertical. Standing straight up, 1.3vessels. Tubesvestibule. The vestibule of the nose is its outer part whichcan be reached by a finger, 11.11virus. The smallest and simplest kind of micro-organism,1.14.9.17virus meningitis. Meningitis due to viruses, 9.17‘Viscup’. A plastic cap put on bottles of blood, 12.9Vitamin B,, A vitamin used for treating some kinds 3fanaemia, 7.7, 7.19volatile. A volatile liquid is one which evaporates v :yeasily, 1.4,2.4volt. A way of measuring the strength of electricitv. Mostcar batteries are 12 volts. Some mains electiicity is110 volts, some is 220 volts, 3.7

Vocabulary Indexvolume. The space that something takes up, occupies orfills, 5.7Wwush bottle (ML 8). A special bottle which lets fluidcome out of a tube when you tip or squeeze it, 2.2wusher. A common kind of washer is a rubber circlewith a hole in it. Washers are often used to stop gasesor liquid escaping. Metal washers are often used withnuts and bolts, 3.4,5.22washing equipment. See 3.12watch glass, polypropq4ene (ML 52). This is a plastic dishor plate, on which chemicals are weighed, 2.2, 5.1water in the laboratory. See 3.2, FIGURE 3- 11water-bath (ML 53). A bath of water kept warm all thetime by electricity or gas, 12.6wedge. Something which is thick at one end and thin atthe other, 5.11white blood cell. A leucocyte, 7.14white cell count. The number of white cells in each cubicmillimetre of blood, 7.22, 7.29white cell dilutingfluid. Fluid used for diluting blood tocount the white cells, 3.45, 7.29, 9.9wick (ML 54). The cloth in a lamp which is wet with oilor spirit, 2.2wound. A cut in the bodyWuchereriu buncrofti. A filarial worm, 7.3 7Wuchereriu muloyi. A filarial worm, 7.37XxyZoZ, xylene (ML 101). This is a volatile liquid used forcleaning the lenses of microscopes, 2.4, 6.16, PictureZ, FIGURE 6-20Yyeasts. Plant-like micro-organisms related to fungi, 8.13,11.15Zzero. Nothing or ‘O’, 1.3Ziehl-Neelsen ‘s method. A way of stainingMycobucterium tuberculosis and MycobacteriumZeprue, 11.1

_.,,:,. I _. , ,( * , ),), OI.,I‘j-‘)I: and use‘our own1 graphHAEMOGLOBIN - GRAMS PER 100 ML - ‘GRAMS%’ Id-6Neutralgrey standardFig. 5-10 Making your own graph for the EEL

- 80A CHARTFOR THEMICROHAEMATOCRIT.’ 10’ 0C

Haemoglobin g%increasinganaemiavery low MCHCr’normalthe dotted line is an example of how touse this nomogram: the patient’s haemoglobinwas 8.1 g % and his haematocrit 34% : a rulerhas been put across the scales, as shown bythe dotted line : it cuts the MCHC scale at24% : the patient’s MCHC is 24%r’Haematocrit % I i80 70 60 50 40 /rlrIlllrRl~ ~11111111 In~rlIrrrIr30 LII I I1 ‘120I I’III’l”‘J’I’15I 110increasingly low haematocrit very lowhaematocritFig. 7-4 A nomogram for the MCI-E

Conversionscales for the Grey Wedge Photometernormal fasting blood sugar20 ( 40 60 i 80 100 120 140 1qo 180 200t:':1:':r:':II':iI!:;:':iI'-I'rI I I 1 I I , I10 30 50 7p 90 110 130 150 170 190 2llpGrey wedge scale 0serum ureaIexample, if the grey wedge scale read 70, the blood sugar would be 105% and the serum urea 190 mgthese areapproximatescalesonlyFig. 7-35The blood urea

“-;

1 2 31. A normal blood film with a neutrophil polymorph at its several segments and there are many very small purpletop left corner. The red cells are nearly all the same size granules in its cytoplasm. 3. An eosinophil polymorphand shape and are well filled with haemoglobin. 2. The with many larger pink granules in its cytoplasm. Thesesame neutrophil polymorph as in Plate 1. Its nucleus has sometimes stain slightly purple, as in this film.4 5 64. A hypochromic microcytic film from a very anaemic 5. A basophil polymorph with a segmented nucleus andpatient with a low MCHC. The cells differ greatly in size big dark blue granules. Part of a monocyte and some(anisocytosis) and shape (poikilocytosis) with many platelets are also shown. 6. A small lymphocyte with asmall cells (microcytes). They have a pale empty centre round nucleus, clear pale blue cytoplasm and fewand are poorly filled with haemoglobin (hypochromic). granules.7 8 97. A normochromic macrocytic blood film. The cells are blue cytoplasm. 9. A monocyte with a kidney-shapedwell filled with haemoglobin (normochromic), there is nucleus, more uneven nuclear chromatin than theanisocytosis and many cells are larger than normal lymphocytes, and some purple granules in a ‘smoky’(macrocytes). 8. A large lymphocyte with a kidney- cytoplasm.shaped nucleus and few purple granules in a clear pale

10 11 12These are fresh unstained wet blood films from donor is also compatible. 12. The cells have agglutinated in anblood being cross-matched (see Pictures 14, 15, and 16, ABO mismatch. Much larger agglutinates were seen inFigure 12-3). 10. The red cells have formed a few small other parts of the film -the blood is incompatible. Somerouleaux (‘piles of coins’) -the blood is compatible. crenated cells, due to slow drying of the blood, are seen11. There are large rouleaux due to ‘Dextran’-the blood in 10 and 11.1314 15These are ail wet films of sickle cells (Section 7. 24). contrast microscope. 15. This blood has also sickled, but13. Several sickle cells with long sharp points or spikes most of the cells have more and shorter spikes. This iscan be seen. 14. These long spikes are seen much better the ‘holly leaf’ kind of sickling. A white cell and twowhen the same slide as 13 is looked at with a phase platelets are also seen.16 1716. A Leishman stained thin blood film from a patient ring (see Picture D, Figure 7-II), and one long pointednrith sickle cell anaemia (haemoglobin SS). There is a sickle cell. 17. Reticulocytes from a cresyl blue stainedate normoblast with a round dark nucleus, some slightly dried blood film. This patient had a severe reticulocytosis>olychromatic (purplish staining) red cells, some target with 300/,, reticulocytes (normal adult less than 2’Y”).:ells with a well stained centre around which is a paler

18 19 20 21Plasmodium vivax. 18 and 19. These are both young that in 18. 20 and 21. These show developing trophotrophozoites.Fine red dots-Schuffner’s dots-can be zoites with irregular amoeboid cytoplasm. In 21 there isseen in the cytoplasn of all infected red cells. The a clear pale vacuole in the trophozoite. Two granules ofinfected cells in 19-26 are enlarged, and also perhaps pigment can be seen in the cytoplasm of 20.22 23 24 25Plasmodium vivax. 22. This is an early schizont, the small pieces all through the cell. 24. A male gametocyte.chromatin has split into three pieces, Schuffner’s dots are The nuclear chromatin is mostly spread through the cell.seen and there is a small clear vacuole. 23. This is a late 25. A female gametocyte in which the chromatin is inschbont which will soon break up into many merozoites. one lump.The nuclear chromatin has already divided into many26 27 28 29Plasmodium malariae. 26 and 27. These are young cytoplasm of the trophozoites, especially 28 and 29. Thetrophozoites. 28 and 29. These are older trophozoites. cytoplasm of P. malariae is often spread out in a bandThe infected red cells are not enlarged and there are no across the cell. as in 26 and 28.Schuffner’s dots. There is some black pigment in the

30 31 32 33Plasmodium malariae. 30. The lowest parasite to the schizont with chromatin broken into pieces, also anright is probably an early gametocyte, and the other two early trophozoite with a large chromatin dot. 32. A maleearly schizonts. The infected cells are not enlarged. As gametocyte. 33. A female gametocyte which is largerusual with P. malariae there is much black pigment and and its chromatin more together in one lump.the uninfected part of the cell looks normal. 31. A later34 35 36 37Plasmodium falciparum. 34 and 35. These show heavy in the cytoplasm of the red cell-Maurer’s spots. Theseinfections with young trophozoites. Several cells have are larger and scarcer than Schuffner’s dots, and aremore than one parasite. Several parasites have a double only seen in cells infected with the older trophozoites ofchromatin dot. 36. Two trophozoites are stuck to the P. falciparum.edge of a red cell. 37. An older trophozoite with red spots38 39 40 41Plasmodium falciparum. 38. Three trophozoites, two of 41. ‘Crescents’ -a female gametocyte (left) and a malethem showing Maurer’s spots. 39. Several young tropho- gametocyte (right). In the female the chromatin iszoites, including two in one cell, also an early schizont together in a lump and the cytoplasm bluish. In the malewith chromatin in three parts. 40. A late schizont with the chromatin is spread out and,the cytoplasm purplish.chromatin in several parts and a large piece of pigment.

PARASITES IN THICK AND THIN BLOOD FILMS PLATES 42-5042 43 4442. Two mature troghozoites of Plasmodium ova/e is also seen in the thick film 46 below. 44. Trypanosomastained by Leishman’s method. The red cells are enlarged rhodesierke or T. gambiense. These two trypanosomesand usually have an irregular oval shape like those shown look the same in a blood film. Several of the red cellshere. Schuffner’s dots are easily seen. 43. Borrelia duttoni are very polychromatic (purple staining).stained with methylene blue. This snake-like bacterium45 46 4745. This is a Giemsa thick film showing two gametocytes infection with many young malarial trophozoites, probofP. falciparum (‘crescents’), many young trophozoites, ably P. f.%kiparum. All the haemoglobin should be washedprobably also P. falciparum. and two polymorphs. out of a Giemsa thick film, and it should look like 45.46. A thick film stained by Field’s method showing Here too much remains and makes the film yellow-green.Borrelia duttoni. 47. A Giemsa thick film showing a heavy48 49 5048. Trypanosomes in a thick film stained by Field’s debris shown here. You may have to search hard to findmethod. The clear orange background of haemoglobin a parasite like that at the bottom of the plate-not allfrom the lysed cells lets the trypanosomes be seen very films are as easy as the next one. 50. A Field’s thick filmeasily. 49. This is another Field’s thick film and is not showing many young trophozoites-probably P. falciwellstained. Try to get films like 48 and avoid the brown parum.

51 and 52. These are the same species of microfilaria. with this species. It is thus Dipetalonema perstans, which51 is a Giemsa stained thick film with much dark purple is also called Acanthocheilonema perstans. Somethingbackground. 52 is a Leishman stained thin blood film. else is seen in this film. What is it? Answer undernea:hThis microfilaria has no sheath and nuclei go right to the Plate 95.tip of its tail which is rounded in a small knob, as is usual53A 53853A. This is a Giemsa stained thick blood film. 536. This the tips of their tails. They are Loa loa -see Figure 7-31.is an iron haematoxylin stained film showing the tails of It may not be easy to tell the species of a microtilaria.two microfilariae. The microfilariae in both plates are Look at several. Look for a sheath, and then at the nucleicovered with sheaths and there are many nuclei right to at the tip of the tail.54 55A 55B54. This is a Giemsa thick film of Brugia malayi showing nuclei stop well before the ends of their sharply pointedthe sheath and two nuclei in the tail. 55A and 558. These tails. They are both probably the microfilariae of W.show a microfilaria and the tail of another from the same bancrofti which have lost their sheaths.Giemsa film. Neither microfilaria has a sheath and their

56 57All the plates on this page are centrifuged urinary deposits. them joined together. These are normal, and in womenThey are colourless specimens of poor contrast, so the often come from the vagina. 57. Many very small bacteria,iris diaphragm of the microscope was nearly closed to some round ball-like pus cells, and three red cells. Allincrease the contrast, and they were photographed in these things are abnormal.little light. 56. Some flattened epithelial cells, several of58 59 6058. Two mycelia (‘?hreads’) of a mould or fungus, and cells just below this branch. 59. Some ‘envelope shaped’some pus cells. The right hand mycelium branches near oxalate crystals. 60. A hyaline (‘glass-like’) cast can bethe bottom, and a short branch to the left is starting to seen going from the top to the bottom of the centre ofgrow half way up. A cell wall can be seen dividing two the plate. It contains a few granules.61 62 63 64All the plates in this row show various kinds of cast. granular cast. Many pus cells can also be seen in these61. A low power view of a cellular cast. 62. A high power plates. Another important kind of cast which is notview of the same cast as 61 showing the cells more shown is that made from red cells.clearly. 63. Another kind of -cellular cast. 64. A small

65 66 67All the ova on this page are unstained, except for 70,74, one cell, but its nucleus has already divided into three.and 75, which have been stained red with merthiolate 66. The hookworm ova is now in the morula (‘ball’)solution (MIF). 65-69 show stages in the life of the hook- stage with about eight cells. 67. This is a later morulaworm, Ancylostoma duodenale or Necator americanus with too many cells to count.which have ova that look the same. 65. There is still only68 69 70 7168. A hookworm embryo which is about to hatch and 10-7). 70. The ‘tea tray’ ovum of the whipworm,become a larva. 69. A newly hatched larva. This might Trichuris trichiura. All the rest of the ova on this page,be a hookworm, and it might be the larva of Strongyloides. 71-75, are different kinds of Ascaris. 71. The thick roughIt is not possible to tell the difference without looking outside of an Ascaris ovum.more closely at its mouth and tail (Plate 82 and Figure72 73 74. 7572. Most Ascaris ova have a thick rough outer wall or ticate. 75. Most Ascaris ova are fertile and come fromcortex like 71. Sometimes they lose part of this cortex female worms that have mated with males. Some ova areand become ‘decorticate’ like this ovum which is in the infertile, like this one. Infertile Ascaris ova are larger,four celled morula stage. 73. Another decorticate Ascaris longer, and thinner walled with more granular cytoplasmovum. 74. Another Ascaris ovum which is partly decor- than fertile ova.

76 77 78This row of plates show the three different kinds of at one side which is hard to see, and is not shown onSchistosome ova. 76. This is Schistosoma haematobium this plate. If the embryo (young worm) is alive inside awith a terminal (end) spine. 77. Schistosoma mansoni Schistosome ovum, small hairs or cilia can often be seenwith a lateral (side) spine. 78. Schistosoma japonicum waving about on its surface.is smaller and rounder than the others with a small spine79 80 81 82The ova of the tape worms Taenia solium and Taenia ovum of the dwarf (small) tapeworm, Hymenolepis nana.saginatalookthesame,so thattheovumin79and80might Three pairs ot hooks can be seen inside 80 and 81.have come from either of these worms. 79. A Taenia ovum 82. The larva of Strongyloides stercoralis showing itslooked at from on top showing the rough outer surface. short mouth.80. The same ovum looked at through the middle. 81. The28384 8583. This is the ovum of Fasciolopsis boski. The oper- which are not easy to tell from one another. 85. This isculum (‘door’) of this large ovum is not easy to see, but a ‘Sellotape’ swab from the edge of the anus showingit is probably at the bottom of the plate. 84. This,may be the ova of Enterobius vermicularis-see Figure 1 O-6.any one of several much smaller ova, Heterophyes hetero- Notice how these ova are flattened on one side.phyes, Clonorchis sine&s, or Metagonimus yokogawai,

,;^3ROTOZOA. ETC. IN THE STOOLS PLATES 86-9586 87 88 896. This is a low power view of E. bisto/vtica showing cyst of E. co/i in iodine. 88 shows the typical E. co/iseudopodia (‘feet’) and ingested (‘eaten’) red cells. nucleus (see Figure IO-lo), also some thin sharp,7. A high power view of E. histolytica in red eosin chromidial bars. Two nuclei are shown in 88 and five inolution showing ingested red cells and a pseudopodium 89, but more were seen above and below the planef clear ectoplasm. 88. A cyst of E. coli in saline. 89. A (level) of the plate.90 91 92 93All the plates in the row show high power views of the A curved bar is well seen. 93. This shows the axostylecysts of Giardia la.nblia. The cysts in 90 are stained with and the clear space inside the cyst wall (see Figureiodine, while the others are red MIF specimens. 90. The 10-13). If you cannot identify moving flagellates in thecurved bars are well shown. 91. Two nuclei can be seen. stool, look for these cysts.9%. This is the same cyst as 91, but at a different level.94 9594. A meat fibre. Note its brown colour, its rounded patient with bacillary dysentery (see Picture A, Figureshape from having been partly digested, and the cross 10-I 2). (Answer to the question under Plate 52. Therestriations (lines or stripes) going across it. 95. A low is a trypanosome at the top right hand corner of the plate.)power view of red cells and pus cells in the stool of a

96 97Both these plates show Gram stained CSF films from two Gram-negative nuclei of polymorphs are also seen,patients with pneumococcal meningitis. 96. This is a high 97. This shows a severe infection with many bacteriapower view showing purple staining Gram-positive and few polymorphs. It is likely that the patient is beingcocci -Streptococcus pneumoniae (pneumococci) - overcome by his infection.lying end to end (see Figure 9-6). The red staining9898. This is a Gram stained film of the CSF from a patient shaped) bacilli. Some are long and others are short likewith Haemophilus influenzae meningitis. It shows two cocci. These are Haemophilus influenzae -see Figurepartly destroyed polymorphs and some red staining 9 -6, and Sectlon 9. 16.Gram-negative bacteria. These are pleomorphic (many99 100These are both wet films of the sputum showing ova of ova and many pus cells. 100. A high power view of onethe trematode worm Paragonimus westermani. The adult of the ova in 09, The operculum ,(‘ddor’) is not y[ellworms are in the lungs and their ova have been coughed seen, but is probably at the top of the ovum in thisup in the sputum. 99. A low power view showing five plate-see Picture D, Figure 10 -9.

101 102101. This shows red staining Gram-negative cocci lying side by side. 102. This shows clumps (groups) of purpleinside some of the polymorphs in the pus from a urethral staining Gram-poiitive cocci in a film of pus taken fromdischarge. The patient has gonorrhoe?, and these are a septic (infected) wound. These are probably Staphylo-Neisseria gonorrhoeae, or gonococci. A fe.v cocci are cocci. Many red staining Gram-negative pus cells are alsoseen outside the cells as diplococci (double cocci) lying seen.103 104103. Sputum from a patient with tuberculosis stained by stained smear of a nasal scraping from a patient withthe Ziehl-Neelsen method to show red acid and alcohol lepromatous leprosy showing red staining AAFBfastbacilli (‘AAFB’) -Mycobacterium tuberculosis. The Mycobacterium leprae-in globi (groups inside the cell).cells in the sputum have been stained green with mala- This is a lower powered view than 104 and single bacillichite green -see Section 11. 1. 104. A Ziehl-Neelsen are not seen so easily.105 106 107These are all Ziehl-Neelsen stained films from leprosy now mostly non-solids, and the morphological index ispatients. 105. Most bacilli are solid, dark, uniformly low. 107. After more treatment the bacilli have broken upstaining red rods. They are ‘solids’ and the morphological further. There is now only acid fast debris and a few nonindexis high. 106. The patient has been treated and paler solids left.staining bacilli are breaking up into granules-they are